Chimeric antigen receptors based on alternative signal 1 domains

ABSTRACT

Described herein are methods for producing and utilizing an alternative signal 1 domain to construct an optimally signaling CAR. Alternative signal 1 domains of the present technology are based on alternatives to CD3ζ, including mutated ITAMs from CD3ζ (which contains 3 IT AM motifs), truncations of CD3ζ, and alternative splice variants known as CD3s, CD3 theta, and artificial constructs engineered to express fusions between CD3s or CD30 and CD3ζ. CAR polypeptides comprising alternative signal 1 domains are utilized to engineer CAR T cells. Further, this technology related to methods of treating cancer by administering to a subject in need thereof CAR T cells comprising alternative signal 1 domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational PCT application PCT/US2018/013213, filed Jan. 10, 2018,which claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Nos. 62/444,605, filed Jan. 10, 2017, 62/472,275, filed Mar.16, 2017, 62/580,243, filed Nov. 1, 2017, and 62/584,060, filed Nov. 9,2017, the contents of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

-   -   The instant application contains a Sequence Listing which has        been submitted electronically in ASCII format and is hereby        incorporated by reference in its entirety. Said ASCII copy,        created Jun. 18, 2019, is named        51295-004004_Sequence_Listing_6.18.19_ST25 and is 76,888 bytes        in size.

TECHNICAL FIELD

The technology described herein relates to immunotherapy.

BACKGROUND

Chimeric antigen receptor (CARs) provide a way to direct a cytotoxic Tcell response to target cells expressing a selected target antigen, mostoften a tumor antigen or tumor-associated antigen. CARs are anadaptation of the T cell receptor, where the antigen binding domain isreplaced with the antigen binding domain of an antibody thatspecifically binds the derived target antigen. Engagement of the targetantigen on the surface of a target cell by a CAR expressed on a T cell(“CAR T cell” or “CAR-T”) promotes killing of the target cell.

SUMMARY

Chimeric antigen receptors (CARs) generally include the full length CD3ζ(CD zeta) signaling domain. CD3ζ is thought to mediate “signal 1” in Tcell activation. However, these signaling domains are phosphorylatedupon T cell activation and have been associated with proliferation,cytokine secretion, cytotoxicity, and activation-induced cell death. Asdescribed herein, the present technology utilizes alternative signalingdomains that decouple the cytotoxic signal from the activation-inducedcell death signal which is mediated by engagement of the CAR andactivation of CD3ζ. This change provides CAR-alternative T cells whichdisplay unexpectedly higher potency as compared to CAR-original T cells.The technology described herein is directed in part to the surprisingfinding that alternative signal 1 domains result in more efficientsignaling of a CAR T cell.

Accordingly, one aspect of the technology described herein relates to achimeric antigen receptor (CAR) polypeptide comprising; an extracellulardomain comprising a target-binding sequence, a transmembrane domain, anintracellular domain of a co-stimulatory molecule, and a T cellintracellular signaling domain that lacks a functional ITAM3 sequence.

Another aspect of the technology described herein relates to a CARpolypeptide comprising an extracellular domain comprising a B cellmaturation antigen (BCMA)-binding sequence, a transmembrane domain ofCD8, an intracellular domain of 4-1BB, and a T cell intracellularsignaling domain that lacks a functional ITAM3 sequence.

Another aspect of the technology described herein relates to a CARpolypeptide comprising an extracellular domain comprising a CD37-bindingsequence, a transmembrane domain of CD8, an intracellular domain of4-1BB, and a T cell intracellular signaling domain that lacks afunctional ITAM3 sequence.

In one embodiment of any aspect, the transmembrane domain is thetransmembrane domain of CD8 or 4-1BB.

In one embodiment of any aspect, the intracellular signaling domaincomprises a CD3 ITAM3 sequence selected from SEQ ID NOs: 13, 14, 33, or34.

In one embodiment of any aspect, the intracellular signaling domaincomprises a CD3 ITAM3 sequence comprising a deletion relative to theCD3ζ ITAM3 sequence of SEQ ID NO: 13.

In one embodiment of any aspect, the T-cell intracellular signalingdomain is the intracellular signaling domain of CD3eta (CD3ε) or CD3theta (CD3θ).

In one embodiment of any aspect, the target-binding sequence comprises aligand of the target or an antibody reagent that specifically binds thetarget. In one embodiment, the target is BCMA. In one embodiment, thetarget is CD37.

In one embodiment of any aspect, the antibody reagent comprises an scFv.

In one embodiment, the antibody reagent has a sequence selected from SEQID NOS: 1, 5, or 9.

In one embodiment of any aspect, the BCMA-binding sequence comprises aligand of BCMA or an antibody reagent that specifically binds BCMA. Inone embodiment, the CD37-binding sequence comprises a ligand of CD37 oran antibody reagent that specifically binds BCMA.

Another aspect of the technology described herein relates to a mammaliancell comprising any of the CAR polypeptides described herein, or anucleic acid encoding any of the CAR polypeptides as described herein.

In one embodiment of any aspect, the cell is a T cell. In oneembodiment, the cell is a human cell. In one embodiment, the cell isobtained from an individual having or diagnosed as having cancer, aplasma cell disorder, or autoimmune disease.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: engineering a T cell toexpress any of the CAR polypeptides described herein on the T cellsurface; and administering the engineered CAR T cell to the subject.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising administering a mammaliancell comprising a CAR polypeptide as described herein.

In one embodiment of any aspect, the cancer is a CD37+ or BCMA+ cancer.In one embodiment, the CD37+ cancer is lymphoma or leukemia. In oneembodiment, lymphoma is B-cell Non-Hodgkin Lymphoma (NHL), mantle celllymphoma, Burkitt's lymphoma, B cell lymphoblastic lymphoma or T celllymphoma (e.g., peripheral T cell lymphoma (PTCL), including cutaneousT-cell lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)). Inone embodiment, the leukemia is acute myeloid leukemia (AML).

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: engineering a T cell tocomprise a CAR polypeptide as described herein on the T cell surface,and administering the engineered CAR T cell to the subject, wherein thesubject is non-responsive to anti-CD19 and/or anti-CD20 therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising administering a mammaliancells comprising a CAR polypeptide as described herein to the subject,wherein the cell comprises a CAR polypeptide comprising an extracellulardomain comprising a CD37-binding sequences, and wherein the subject isnon-responsive to anti-CD19 and/or anti-CD20 therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising selecting a subject whois non-responsive to anti-CD19 and/or anti-CD20 therapy, engineering a Tcell to comprise a CAR polypeptide as described herein on the T cellsurface, and administering the engineered CAR T cell to the subject.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising selecting a subject whois non-responsive to anti-CD19 and/or anti-CD20 therapy, administering amammalian cell comprising a CAR polypeptide as described herein to thesubject, wherein the cell comprises a CAR polypeptide comprises anextracellular domain comprising a CD37-binding sequence.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising engineering a T cell tocomprise a CAR polypeptide as described herein on the T cell surface,administering the engineered CAR T cell to the subject, wherein thesubject is concurrently administered an anti-CD19 and/or anti-CD20therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising administering a mammaliancell comprising a CAR polypeptide as described herein to the subject,wherein the cell comprises a CAR polypeptide comprising an extracellulardomain comprising a CD37-binding sequence, and wherein the subject isconcurrently administered an anti-CD19 and/or anti-CD20 therapy.

Another aspect of the technology described herein relates to acomposition comprising a CAR T cell as described herein, formulated forthe treatment of cancer. In one embodiment, the composition furthercomprises a pharmaceutically acceptable carrier.

Definitions

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed technology, because the scope of thetechnology is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thistechnology belongs. If there is an apparent discrepancy between theusage of a term in the art and its definition provided herein, thedefinition provided within the specification shall prevail.

Definitions of common terms in immunology and molecular biology can befound in The Merck Manual of Diagnosis and Therapy, 19th Edition,published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular CellBiology and Molecular Medicine, published by Blackwell Science Ltd.,1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Janeway's Immunobiology, KennethMurphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014(ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green andJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2012) (ISBN 1936113414); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS),John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and CurrentProtocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David HMargulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons,Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which areall incorporated by reference herein in their entireties.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. Where applicable, adecrease can be preferably down to a level accepted as within the rangeof normal for an individual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, an “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include, for example, chimpanzees, cynomologousmonkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include,for example, mice, rats, woodchucks, ferrets, rabbits and hamsters.Domestic and game animals include, for example, cows, horses, pigs,deer, bison, buffalo, feline species, e.g., domestic cat, caninespecies, e.g., dog, fox, wolf, avian species, e.g., chicken, emu,ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments,the subject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,” “patient” and “subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of diseasee.g., cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. leukemia or another type of cancer, among others) or one or morecomplications related to such a condition, and optionally, have alreadyundergone treatment for the condition or the one or more complicationsrelated to the condition. Alternatively, a subject can also be one whohas not been previously diagnosed as having such condition or relatedcomplications. For example, a subject can be one who exhibits one ormore risk factors for the condition or one or more complications relatedto the condition or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

A “disease” is a state of health of an animal, for example a human,wherein the animal cannot maintain homeostasis, and wherein if thedisease is not ameliorated, then the animal's health continues todeteriorate. In contrast, a “disorder” in an animal is a state of healthin which the animal is able to maintain homeostasis, but in which theanimal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

As used herein, the terms “tumor antigen” and “cancer antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), and fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses. Many tumor antigens have been defined in terms ofmultiple solid tumors: MAGE 1, 2, & 3, defined by immunity;MART-1/Melan-A, gp100, carcinoembryonic antigen (CEA), HER2, mucins(i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acidphosphatase (PAP). In addition, viral proteins such as some encoded byhepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) havebeen shown to be important in the development of hepatocellularcarcinoma, lymphoma, and cervical cancer, respectively.

As used herein, the term “chimeric” refers to the product of the fusionof portions of at least two or more different polynucleotide molecules.In one embodiment, the term “chimeric” refers to a gene expressionelement produced through the manipulation of known elements or otherpolynucleotide molecules

In some embodiments, “activation” can refer to the state of a T cellthat has been sufficiently stimulated to induce detectable cellularproliferation. In some embodiments activation can refer to inducedcytokine production. In other embodiments, activation can refer todetectable effector functions. At a minimum, an “activated T cell” asused herein is a proliferative T cell.

As used herein, the terms “specific binding” and “specifically binds”refer to a physical interaction between two molecules, compounds, cellsand/or particles wherein the first entity binds to the second, target,entity with greater specificity and affinity than it binds to a thirdentity which is a non-target. In some embodiments, specific binding canrefer to an affinity of the first entity for the second target, entity,which is at least 10 times, at least 50 times, at least 100 times, atleast 500 times, at least 1000 times or more greater than the affinityfor the third nontarget entity under the same conditions. A reagentspecific for a given target is one that exhibits specific binding forthat target under the conditions of the assay being utilized. Anon-limiting example includes an antibody, or a ligand, which recognizesand binds with a cognate binding partner (for example, a stimulatoryand/or costimulatory molecule present on a T cell) protein.

A “stimulatory ligand,” as used herein, refers to a ligand that whenpresent on an antigen presenting cell (APC e.g., a macrophage, adendritic cell, a B-cell, an artificial APC, and the like) canspecifically bind with a cognate binding partner (referred to herein asa “stimulatory molecule” or “co-stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, proliferation, activation, initiation of an immune response,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an APC that specifically binds a cognate co-stimulatory molecule on aT cell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation, activation, differentiation, and thelike. A co-stimulatory ligand can include, but is not limited to,4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducibleCOStimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that bindsToll-like receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also can include, but is not limited to, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD3θ,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds withCD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA, a Toll-like receptor, CD27,CD28, 4-1BB, OX40, CD3θ, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and CD83.

In one embodiment, the term “engineered” and its grammatical equivalentsas used herein can refer to one or more human-designed alterations of anucleic acid, e.g., the nucleic acid within an organism's genome. Inanother embodiment, engineered can refer to alterations, additions,and/or deletion of genes. An “engineered cell” can refer to a cell withan added, deleted and/or altered gene. The term “cell” or “engineeredcell” and their grammatical equivalents as used herein can refer to acell of human or non-human animal origin.

As used herein, the term “operably linked” refers to a firstpolynucleotide molecule, such as a promoter, connected with a secondtranscribable polynucleotide molecule, such as a gene of interest, wherethe polynucleotide molecules are so arranged that the firstpolynucleotide molecule affects the function of the secondpolynucleotide molecule. The two polynucleotide molecules may or may notbe part of a single contiguous polynucleotide molecule and may or maynot be adjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of ordinary skill willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters asingle amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid and retains the desired activity of the polypeptide. Suchconservatively modified variants are in addition to and do not excludepolymorphic variants, interspecies homologs, and alleles consistent withthe disclosure.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g.ligand-mediated receptor activity and specificity of a native orreference polypeptide is retained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C),Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys(K), Arg (R), His (H). Alternatively, naturally occurring residues canbe divided into groups based on common side-chain properties: (1)hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutralhydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic:His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; (6) aromatic:Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging amember of one of these classes for another class. Particularconservative substitutions include, for example; Ala into Gly or intoSer; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser;Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn orinto Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys intoArg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe intoMet, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyrinto Trp; and/or Phe into Val, into Ile or into Leu.

In some embodiments, a polypeptide described herein (or a nucleic acidencoding such a polypeptide) can be a functional fragment of one of theamino acid sequences described herein. As used herein, a “functionalfragment” is a fragment or segment of a peptide which retains at least50% of the wildtype reference polypeptide's activity according to anassay known in the art or described below herein. A functional fragmentcan comprise conservative substitutions of the sequences disclosedherein.

In some embodiments, a polypeptide described herein can be a variant ofa polypeptide or molecule as described herein. In some embodiments, thevariant is a conservatively modified variant. Conservative substitutionvariants can be obtained by mutations of native nucleotide sequences,for example. A “variant,” as referred to herein, is a polypeptidesubstantially homologous to a native or reference polypeptide, but whichhas an amino acid sequence different from that of the native orreference polypeptide because of one or a plurality of deletions,insertions or substitutions. Variant polypeptide-encoding DNA sequencesencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to a native or reference DNAsequence, but that encode a variant protein or fragment thereof thatretains activity of the non-variant polypeptide. A wide variety ofPCR-based site-specific mutagenesis approaches are known in the art andcan be applied by the ordinarily skilled artisan.

A variant amino acid or DNA sequence can be at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, identical to a native orreference sequence. The degree of homology (percent identity) between anative and a mutant sequence can be determined, for example, bycomparing the two sequences using freely available computer programscommonly employed for this purpose on the world wide web (e.g. BLASTp orBLASTn with default settings).

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites permitting ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare well established and include, for example, those disclosed by Walderet al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik(BioTechniques, Jan. 1985, 12-19); Smith et al. (Genetic Engineering:Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos.4,518,584 and 4,737,462, which are herein incorporated by reference intheir entireties. Any cysteine residue not involved in maintaining theproper conformation of a polypeptide also can be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant crosslinking. Conversely, cysteine bond(s) can be addedto a polypeptide to improve its stability or facilitate oligomerization.

As used herein, the term “DNA” is defined as deoxyribonucleic acid. Theterm “polynucleotide” is used herein interchangeably with “nucleic acid”to indicate a polymer of nucleosides. Typically a polynucleotide iscomposed of nucleosides that are naturally found in DNA or RNA (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine) joined byphosphodiester bonds. However the term encompasses molecules comprisingnucleosides or nucleoside analogs containing chemically or biologicallymodified bases, modified backbones, etc., whether or not found innaturally occurring nucleic acids, and such molecules may be preferredfor certain applications. Where this application refers to apolynucleotide it is understood that both DNA, RNA, and in each caseboth single- and double-stranded forms (and complements of eachsingle-stranded molecule) are provided. “Polynucleotide sequence” asused herein can refer to the polynucleotide material itself and/or tothe sequence information (i.e. the succession of letters used asabbreviations for bases) that biochemically characterizes a specificnucleic acid. A polynucleotide sequence presented herein is presented ina 5′ to 3′ direction unless otherwise indicated.

The term “polypeptide” as used herein refers to a polymer of aminoacids. The terms “protein” and “polypeptide” are used interchangeablyherein. A peptide is a relatively short polypeptide, typically betweenabout 2 and 60 amino acids in length. Polypeptides used herein typicallycontain amino acids such as the 20 L-amino acids that are most commonlyfound in proteins. However, other amino acids and/or amino acid analogsknown in the art can be used. One or more of the amino acids in apolypeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a fatty acidgroup, a linker for conjugation, functionalization, etc. A polypeptidethat has a nonpolypeptide moiety covalently or noncovalently associatedtherewith is still considered a “polypeptide.” Exemplary modificationsinclude glycosylation and palmitoylation. Polypeptides can be purifiedfrom natural sources, produced using recombinant DNA technology orsynthesized through chemical means such as conventional solid phasepeptide synthesis, etc. The term “polypeptide sequence” or “amino acidsequence” as used herein can refer to the polypeptide material itselfand/or to the sequence information (i.e., the succession of letters orthree letter codes used as abbreviations for amino acid names) thatbiochemically characterizes a polypeptide. A polypeptide sequencepresented herein is presented in an N-terminal to C-terminal directionunless otherwise indicated.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein (e.g. a CAR polypeptide) is comprised by a vector. In some of theaspects described herein, a nucleic acid sequence encoding a givenpolypeptide as described herein, or any module thereof, is operablylinked to a vector. The term “vector”, as used herein, refers to anucleic acid construct designed for delivery to a host cell or fortransfer between different host cells. As used herein, a vector can beviral or non-viral. The term “vector” encompasses any genetic elementthat is capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, artificial chromosome, virus,virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain a nucleic acid encoding a polypeptide as described herein inplace of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring nucleic acids into cells eitherin vitro or in vivo. Numerous forms of viral vectors are known in theart.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra-chromosomal DNAthereby eliminating potential effects of chromosomal integration.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. acute lymphoblastic leukemia or other cancer, disease, ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some embodimentsof any of the aspects, a pharmaceutically acceptable carrier can be acarrier other than water. In some embodiments of any of the aspects, apharmaceutically acceptable carrier can be a cream, emulsion, gel,liposome, nanoparticle, and/or ointment. In some embodiments of any ofthe aspects, a pharmaceutically acceptable carrier can be an artificialor engineered carrier, e.g., a carrier in which the active ingredientwould not be found to occur in nature.

As used herein, the term “administering,” refers to the placement of atherapeutic or pharmaceutical composition as disclosed herein into asubject by a method or route which results in at least partial deliveryof the agent at a desired site. Pharmaceutical compositions comprisingagents as disclosed herein can be administered by any appropriate routewhich results in an effective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the technology.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

Other terms are defined within the description of the various aspectsand embodiments of the technology of the following.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic diagram of the generic configuration of aBCMA-targeted CAR. The lentiviral vector construct contains asingle-chain variable region derived from a BCMA-specific monoclonalantibody linked to a CD8 transmembrane domain (CD8 TM), followed by theintracellular domain of 4-1BB (4-1BB ICD, CD137) and the CD3ζ moduleshowing the three putative ITAMs (I, II, III).

FIGS. 2A-2C demonstrate that primary human T cells transduced with ananti-BCMA CAR have anti-myeloma cytotoxic activity in vitro.BCMA-specific CAR T cells lyse target myeloma cells. FIG. 2Ademonstrates BCMA expression in bone marrow cells from a multiplemyeloma patient (bottom histogram) and in a multiple myeloma cell line(RPMI 8226, middle histogram). FIG. 2B depicts results of experiments inwhich CAR T and multiple myeloma (RPMI 8226) cells (GFP+) wereco-cultured in the presence of propidium iodide at a 1:1 effector:target(E:T) ratio. Images were taken at 0 and 24 hours using an EVOS® FLfluorescence imager. FIG. 2C depicts specific cell lysis of multiplemyeloma measured by end-point luminescence at different effector:targetcell ratios after 16-hour co-culture. UTD=Untransduced cells.

FIG. 3 depicts a graph of results obtained with a xenograft model ofmultiple myeloma described herein. Assessment of tumor growth byintra-vital bioluminescence (BLI) detection using aluciferase-expressing multiple myeloma cell line (U266). BLI wasevaluated at increasing time points in immunodeficient mice that wereco-injected with CAR T cells (dark gray line) or left untreated (lightgray line) using an IVIS® Spectrum Imaging System.

FIG. 4 depicts schematic diagrams of an anti-BCMA CAR construct.

FIG. 5 depicts schematic CAR construct diagrams of alternativeintracellular domains.

FIG. 6 depicts a diagram of the experimental approach and a graph of thespecific activation of the indicated CARs.

FIG. 7 depicts a graph of the cytotoxicity of the indicated CAR Ts.

FIG. 8 depicts a graph of CAR-specific cytotoxicity for the indicatedanti-BCMA-CAR configurations

FIG. 9 depicts graphs of cytokine production by the indicated CAR Ts.

FIG. 10 depicts a graph of CAR-specific proliferation for the indicatedCAR Ts. CAR T cells are stimulated every seven days with K562-BCMA+cells. CAR Ts are counted every two days.

FIG. 11 depicts a graph of CD69 activation in CAR T cells bearing theindicated anti-BCMA CAR construct.

FIG. 12 depicts the results of a CD107a degranulation assay.

FIG. 13 demonstrates that CAR-specific activation is independent ofendogenous ITAMs.

FIG. 14 depicts results of in vivo assays with the indicated CAR Ts.

FIG. 15 depicts peripheral blood analysis of CAR T survival capacity.

FIG. 16 depicts graphs demonstrating that inactivation or absence ofITAM3 Generates CAR T Cells with a phenotype associated with augmentedsurvival capacity.

FIG. 17 depicts a schematic diagram of CD3 isoforms.

FIG. 18 depicts a graph of longitudinal analysis of tumor burden in NSGmice infused with anti-BCMA CAR T cells.

FIG. 19 demonstrates transduction efficiency in primary T cells (ND23).

FIG. 20 depicts schematic diagrams of alternative CARs. Darker barswithin CD3 domains indicate ITAMS (2 tyrosine residues per ITAM). *Indicate tyrosine to phenylalanine (Y>F) point mutations. T2A andmCherry not shown.

FIGS. 21A and 21B depict graphs of activation and in vitro cytotoxicityof the indicated CAR Ts.

FIG. 22 depicts a schematic diagram of 4-1BBSTOP CAR. CD3ζ was removedfrom the CAR, instead of merely mutating ITAMs because of other basicrich sequences present within zeta.

FIG. 23 depicts lentivirus titration with SupT1 cells.

FIG. 24 depicts graphs of transduction efficiency in Jurkat T cells.

FIG. 25 depicts a graph of activation assays conducted with anti-BCMAexpressing Jurkat T cells. Results shown are from two independentexperiments.

FIG. 26 depicts graphs of transduction efficiency in ND23 primary Tcells.

FIG. 27 depicts a graph of tumor killing activity of the indicated CAR Tcells. T cells are ND19 T cells.

FIG. 28 depicts a graph of cell proliferation for the indicated ND23anti-BCMA CAR T cells.

FIG. 29 depicts a graph of cell proliferation for the indicated ND19anti-BCMA CAR T cells.

FIG. 30 depicts a schematic of the experimental approach used for invivo tumor assays.

FIG. 31 depicts images of tumor burden in mice at the indicated timepoints.

FIG. 32 depicts a graph of the tumor burden in mice at the indicatedtime points.

FIG. 33 depicts graphs of analysis of blood samples taken from mice 64days after CAR T infusion.

FIG. 34 depicts a graph of CD37 expression in the indicated cells.

FIG. 35 depicts schematic diagrams of anti-CD37 CARs.

FIG. 36 depicts graphs of anti-CD37 CAR expression

FIG. 37 depicts graphs of expansion of anti-CD37 CAR T cells.

FIG. 38 depicts graphs of a cytotoxicity assay demonstrating thatanti-CD37 CAR T cells lyse CD37 positive T cells.

FIG. 39 depicts graphs of cytotoxicity assays and expression analysis oftarget clones.

FIG. 40 depicts graphs of cell proliferation following stimulation ofanti-CD37 CAR T cells with CD37. Assay was begun on day 17 and FACS wasgated for CAR+.

FIG. 41 depicts a schematic of experimental design for anti-CD37 CAR Tcells engraftment and tumor clearance in NSG mice.

FIG. 42 depicts schematics of CAR design. pMGH69 (top construct): antiCD37scFv with light-heavy chains configuration. pMGH70 (bottomconstruct): anti CD37scFv with heavy—light chains configuration.

FIG. 43 depicts graphs of expression of CD37 on cancer cell lines byflow. Jeko-1 (MCL), RAJI (Burkitt lymphoma), and OSU-CLL (CLL) clearlyexpress high level of CD37. NALM6 cells (ALL) do not express CD37.

FIG. 44 demonstrates the generation and expansion of anti-CD37 CAR Tcells. The graph on the left shows the growth curve for anti-CD37-CAR Tcells. Starting at day 0 the T cells are expanded in vitro usingDyna-beads until day 10, then T cell are stimulated with antigen, inthis case with irradiated K562 expressing CD37 and CD19. Graph on theright shows the transduction efficiency of this particular batch of CART cells at day 10 of culture (19.5% and 23.5 of the total cells are CARTs). Bottom left: transduction efficiency of 3 different normal donors.

FIG. 45 demonstrates antigen specific activation. Jurkat-NFAT reportercell line transduced with CD37-CAR constructs is activated in thepresence of target cells (RAJI, OSU-CLL, Jeko-1, NALM-CD37) but not byNALM6 or media alone.

FIG. 46 depicts proliferation capacity of CAR T cells. Proliferationassay: CAR T cells are labeled with CellTrace™ Violet and co-cultured inpresence or absence of antigen. The figure depicts CAR T cells expansionin the presence of CD37+ cells but not with cells negative for CD37.

FIG. 47 demonstrates in vitro tumor cell killing. Cytotoxicity at 16hours of CD37-CAR T, CD19-CAR or control T cells (UTD) when co-culturedat different E:T ratios with tumor cells. Increasing concentration ofeither CD37-CAR T or CAR T-19 led to similar levels of killing of targetcells, while no killing was observed in the control group (UTD).

FIG. 48 demonstrates that cytokine production by CD37-CAR, CD19-CAR orUTD incubated with tumor cells for 16 hrs at 1:1 E:T ratio was analyzedin the culture supernatants by LUMINEX FLEXMAP 3D® assay. Technicalduplicates (N=1 biological).

FIG. 49 depicts an experimental schematic: NSG mice were injected with1e6 tumor cells (Jeko-1 CBG-GFP, i.v.). After 1 week mice wererandomized according to tumor burden and injected with 1e6 (left) or 2e6(right) positive CAR T cells or UTD cells. Mice were imaged every 7 daysand tumor growth was analyzed measuring bioluminescence (represented ingraphs). In both experiment, mice treated with UTD showed diseaseprogression (blue line). CAR T-19 cells are capable of inducingresponses in both conditions.

FIG. 50 demonstrates in vivo CAR efficacy. Representative mice of thesecond experiment are shown in the figure. Presence of CAR T cells inperipheral blood 14 days after T-cell injection. Cells from blood werestained and gated on the expression of human CD3. Percentage of mCherrypositive cells (CAR+) is displayed in the graph.

FIGS. 51A and 51B show CD37 protein expression in normal cells (FIG.51A) and tumor cells (FIG. 51B).

FIG. 52 shows exemplary CAR-37 T cells design.

FIGS. 53A-53D show generation and expansion of CAR-37 T cells.

FIGS. 54A-54C show in vitro cytotoxic activity of CAR-37 T cells.

FIGS. 55A-55C show in vitro cytokine production of CAR-37 T cells.

FIGS. 56A-56C show in vitro effector function of CAR-37 T cells.

FIG. 57 shows expression of CD37 in tumor cells. The top panel showsexpression of CD37 and CD19 in NALM6, CD19 CD38 K562, JEKO-1, RAJI, andOSU-CLL cell lines as assessed by flow cytometry. The bottom panel showsexpression of CD37 and CD17 in MCL patient-derived xenograft (PDX) celllines PDX_44685, PDX_98848, and PDX_96069 as assessed by flow cytometry.The graph on the bottom right panel shows the percent expression of CD19and CD37 in MCL PDX cell lines.

FIGS. 58A-58C demonstrate in vivo efficacy of CAR-37 T cells against MCLPDX tumors. FIG. 58A depicts an experimental schematic. On Day −39, micewere administered 1×10⁶ PDX_98848 MCL PDX cells intravenously. Flowcytometry was performed on days −28 and −14. BLI was performed on Day−1. On day 0, 3×10⁶ UTD (control) or CART positive cells (CAR-37 H-L orCAR-19) were administered intravenously into the mice. Mice were imagedon days 3, 7, 10, 14, 17, 21, and 35, and tumor growth was analyzedmeasuring bioluminescence (FIGS. 58B and 58C). CAR-37 T cells had stronganti-tumor efficacy against MCL PDX tumors in vivo.

FIG. 59A shows expression of CD37 in the peripheral T cell lymphoma(PTCL) cell lines HUT78 (cutaneous T-cell lymphoma (CTCL)) (left panel)and FEPD (anaplastic large cell lymphoma (ALCL)) (right panel) asassessed by flow cytometry.

FIG. 59B shows expression of CD69 in unstimulated (unstim) andstimulated (stim) CAR T cells as assessed by flow cytometry. The leftpanel shows expression of mCherry (CAR+) as assessed by flow cytometry.

FIG. 59C shows the percentage of CD69+ UTD (control) CAR-37 L-H, andCAR-37 H-L cells stimulated with media, FEPD cells, HUT78 cells, orCDR-CD28 beads.

FIG. 60 shows in vitro effector function of UTD (control), CAR-37 L-H,and CAR-37 H-L T cells against HUT78 (CTCL) and FEPD (ALCL) PTCL celllines. The graphs plot percent specific lysis for the indicated E:Tratios.

FIG. 61 shows expression of CD37 in the AML cell lines TF1, MOM13, andTHP1 as assessed by flow cytometry.

DETAILED DESCRIPTION

To date, CARs have generally included a full length CD3ζ signalingdomain. CD3ζ is thought to mediate “signal 1” in T cell activation.However, these signaling domains are phosphorylated upon T cellactivation and have been associated with proliferation, cytokinesecretion, cytotoxicity, and activation-induced cell death. As describedherein, the present technology utilizes alternative signaling domainsthat decouple the cytotoxic signal from the activation-induced celldeath signal mediated by engagement of the CAR and activation of CD3zeta. This change, and others described herein, provide more potentCAR-alternative T cells compared to CAR-original T cells.

Embodiments of the technology described herein relate to the discoverythat including an alternative signal 1 domain in a CAR construct resultsin a more efficient CAR T cell. CAR T cells with alternative signal 1domains exhibit a higher capacity for treating disease, for example,cancer, compared to CAR T cells that utilize the original signal 1domain.

Accordingly, one aspect of the technology described herein relates to aCAR polypeptide comprising: an extracellular domain comprising atarget-binding sequence; a transmembrane domain (including, for example,a transmembrane domain of a co-stimulatory ligand); and a T cellintracellular signaling domain that lacks a functional ITAM3 sequence.

Another aspect of the technology described herein relates to a CARpolypeptide comprising: an extracellular domain comprising aBCMA-binding sequence; a transmembrane domain comprising thetransmembrane domain of CD8 or 4-1BB; and an intracellular domaincomprising a T-cell signaling domain.

Another aspect of the technology described herein relates to a CARpolypeptide comprising: an extracellular domain comprising aCD37-binding sequence; a transmembrane domain comprising thetransmembrane domain of CD8 or 4-1BB; and an intracellular domaincomprising a T-cell signaling domain.

Considerations necessary to make and use these and other aspects of thetechnology are described in the following.

Chimeric Antigen Receptors

The technology described herein provides improved CARs for use inimmunotherapy. The following discusses CARs and the variousimprovements.

It should be understood that while BCMA and CD37 are discussed herein asexamples of CAR targets, they are just that: examples. The principlesdescribed herein with respect to improvements in CAR and therefore CAR Tdesign can be applied to CARs that target any target or antigen,including any cell-surface target or antigen.

The terms “chimeric antigen receptor” or “CAR” or “CARs” as used hereinrefer to engineered T cell receptors, which graft a ligand or antigenspecificity onto T cells (for example naïve T cells, central memory Tcells, effector memory T cells or combinations thereof). CARs are alsoknown as artificial T-cell receptors, chimeric T-cell receptors orchimeric immunoreceptors.

A CAR places a chimeric extracellular target-binding domain thatspecifically binds a target, e.g., a polypeptide, expressed on thesurface of a cell to be targeted for a T cell response onto a constructincluding a transmembrane domain and intracellular domain(s) of a T cellreceptor molecule. In one embodiment, the chimeric extracellulartarget-binding domain comprises the antigen-binding domain(s) of anantibody that specifically binds an antigen expressed on a cell to betargeted for a T cell response. The properties of the intracellularsignaling domain(s) of the CAR can vary as known in the art and asdisclosed herein, but the chimeric target/antigen-binding domains(s)render the receptor sensitive to signaling activation when the chimerictarget/antigen binding domain binds the target/antigen on the surface ofa targeted cell.

With respect to intracellular signaling domains, so-called“first-generation” CARs include those that solely provide CD3zeta (CD3ζ)signals upon antigen binding. So-called “second-generation” CARs includethose that provide both co-stimulation (e.g., CD28 or CD 137) andactivation (CD3ζ) domains, and so-called “third-generation” CARs includethose that provide multiple costimulatory (e.g., CD28 and CD 137)domains and activation domains (e.g., CD3ζ). In various embodiments, theCAR is selected to have high affinity or avidity for thetarget/antigen—for example, antibody-derived target or antigen bindingdomains will generally have higher affinity and/or avidity for thetarget antigen than would a naturally-occurring T cell receptor. Thisproperty, combined with the high specificity one can select for anantibody provides highly specific T cell targeting by CAR T cells.

As used herein, a “CAR T cell” or “CAR-T” refers to a T cell whichexpresses a CAR. When expressed in a T cell, CARs have the ability toredirect T-cell specificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT-cells expressing CARs the ability to recognize an antigen independentof antigen processing, thus bypassing a major mechanism of tumor escape.

As used herein, the term “extracellular target binding domain” refers toa polypeptide found on the outside of the cell which is sufficient tofacilitate binding to a target. The extracellular target binding domainwill specifically bind to its binding partner, i.e. the target. Asnon-limiting examples, the extracellular target-binding domain caninclude an antigen-binding domain of an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner (for example, BCMAor CD37) protein. In this context, a ligand is a molecule which bindsspecifically to a portion of a protein and/or receptor. The cognatebinding partner of a ligand useful in the methods and compositionsdescribed herein can generally be found on the surface of a cell.Ligand:cognate partner binding can result in the alteration of theligand-bearing receptor, or activate a physiological response, forexample, the activation of a signaling pathway. In one embodiment, theligand can be non-native to the genome. Optionally, the ligand has aconserved function across at least two species. In one embodiment, theextracellular target binding domain comprises a non-antibody ligand(e.g., A PRoliferation-Inducing Ligand (APRIL)).

Antibody Reagents

In various embodiments, the CARs described herein comprise an antibodyreagent or an antigen-binding domain thereof as an extracellulartarget-binding domain.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments of any ofthe aspects, an antibody reagent can comprise a monoclonal antibody or apolypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as V_(H)), and a light (L) chainvariable region (abbreviated herein as V_(L)). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody reagent” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, CDRs,and domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, or IgM (as well assubtypes and combinations thereof). Antibodies can be from any source,including mouse, rabbit, pig, rat, and primate (human and non-humanprimate) and primatized antibodies. Antibodies also include midibodies,humanized antibodies, chimeric antibodies, and the like. Fully humanantibody binding domains can be selected, for example, from phagedisplay libraries using methods known to those of ordinary skill in theart.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each V_(H) and V_(L) is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

In one embodiment, the antibody or antibody reagent is not a humanantibody or antibody reagent, (i.e., the antibody or antibody reagent ismouse), but has been humanized. A “humanized antibody or antibodyreagent” refers to a non-human antibody or antibody reagent that hasbeen modified at the protein sequence level to increase its similarityto antibody or antibody reagent variants produced naturally in humans.One approach to humanizing antibodies employs the grafting of murine orother non-human CDRs onto human antibody frameworks.

In one embodiment, a CAR's extracellular target binding domain comprisesor consists essentially of a single-chain Fv (scFv) fragment created byfusing the V_(H) and V_(L) domains of an antibody, generally amonoclonal antibody, via a flexible linker peptide. In variousembodiments, the scFv is fused to a transmembrane domain and to a T cellreceptor intracellular signaling domain, e.g., an engineeredintracellular signaling domain as described herein.

Antibody binding domains and ways to select and clone them are wellknown to those of ordinary skill in the art. In another embodiment, theantibody reagent is an anti-CD37 antibody reagent and has the sequenceselected from SEQ ID NO: 5 or 9. In one embodiment, the anti-OTK3antibody reagent corresponds to the sequence of SEQ ID NO: 5 or 9; orcomprises the sequence of SEQ ID NO: 5 or 9; or comprises a sequencewith at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or greater sequenceidentity to the sequence of SEQ ID NO: 5 or 9.

In another embodiment, the antibody reagent is an anti-BCMA antibodyreagent and has the sequence of SEQ ID NO: 1. In one embodiment, theanti-CD28 antibody corresponds to the sequence of SEQ ID NO: 1; orcomprises the sequence of SEQ ID NO: 1; or comprises a sequence with atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or greater sequence identity tothe sequence of SEQ ID NO: 1.

In one embodiment, the CARs useful in the technology described hereincomprise at least two antigen-specific targeting regions, anextracellular domain, a transmembrane domain, and an intracellularsignaling domain. In such embodiments, the two or more antigen-specifictargeting regions target at least two different antigens and may bearranged in tandem and separated by linker sequences. In anotherembodiment, the CAR is a bispecific CAR. A bispecific CAR is specific totwo different antigens.

Target/Antigen

Any cell-surface moiety can be targeted by a CAR. Most often, the targetwill be a cell-surface polypeptide differentially or preferentiallyexpressed on a cell one wishes to target for a T cell response. In thisregard, tumor antigens or tumor-associated antigens provide attractivetargets, providing a means to target tumor cells while avoiding or atleast limiting collateral damage to non-tumor cells or tissues.Non-limiting examples of tumor antigens or tumor-associated antigensinclude CEA, Immature laminin receptor, TAG-72, HPV E6 and E7, BING-4,Calcium-activated chloride channel 2, Cyclin B1, 9D7, Ep-CAM, EphA3,Her2/neu, Telomerase, Mesotheliun, SAP-1, Survivin, BAGE family, CAGEfamily, GAGE family, MAGE family, SAGE family, XAGE family,NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A/MART-1, Gp100/pme117, Tyrosinase,TRP-1/-2, MC1R, BRCA1/2, CDK4, MART-2, p53, Ras, MUC1, and TGF-βRII.

In one embodiment, the target is B cell maturation antigen (BCMA), alsoknown as tumor necrosis factor receptor superfamily member 17(TNFRSF17). BCMA is a cell surface receptor expressed preferentially onmature B lymphocytes that specifically recognizes B cell activatingfactor (BAFF). BCMA sequences are known for a number of species, e.g.,human BCMA (NCBI Gene ID: 608) polypeptide (e.g., NCBI Ref SeqNP_001183.2) and mRNA (e.g., NCBI Ref Seq NM_001192.2). BCMA can referto human BCMA, including naturally occurring variants, molecules, andalleles thereof. In some embodiments of any of the aspects, e.g., inveterinary applications, BCMA can refer to the BCMA of, e.g., dog, cat,cow, horse, pig, and the like. Homologs and/or orthologs of human BCMAare readily identified for such species by one of skill in the art,e.g., using the NCBI ortholog search function or searching availablesequence data for a given species for sequence similar to a referenceBCMA sequence.

In one embodiment, the BCMA-binding sequence comprises a ligand of BCMAor an antibody reagent that specifically binds BCMA. In one embodiment,the antibody reagent that specifically binds BCMA is a scFv from ahumanized anti-BCMA m murine antibody. The orientation of a humanizedmurine antibody-derived single-chain variable fragment can beV_(L)-V_(H) or V_(H)-V_(L).

In one embodiment, the target is CD37. CD37 is cell surface protein thatcontains four hydrophobic transmembrane domains. CD37 is expressedexclusively on immune cells; CD37 is highly expressed on mature B cells,and moderately expressed on T cells and myeloid cells. CD37 sequencesare known for a number of species, e.g., human CD37 (NCBI Gene ID: 951)polypeptide (e.g., NCBI Ref Seq NP_001035120.1) and mRNA (e.g., NCBI RefSeq NM_001040031.1). CD37 can refer to human CD37, including naturallyoccurring variants, molecules, and alleles thereof. In some embodimentsof any of the aspects, e.g., in veterinary applications, CD37 can referto the CD37 of, e.g., dog, cat, cow, horse, pig, and the like. Homologsand/or orthologs of human CD37 are readily identified for such speciesby one of skill in the art, e.g., using the NCBI ortholog searchfunction or searching available sequence data for a given species forsequence similar to a reference CD37 sequence.

In one embodiment, the CD37-binding sequence comprises a ligand of CD37or an antibody reagent that specifically binds CD37.

Transmembrane Domain

Each CAR as described herein necessarily includes a transmembrane domainthat joins the extracellular target-binding domain to the intracellularsignaling domain.

As used herein, “transmembrane domain” (TM domain) refers to thegenerally hydrophobic region of the CAR which crosses the plasmamembrane of a cell. The TM domain can be the transmembrane region orfragment thereof of a transmembrane protein (for example a Type Itransmembrane protein or other transmembrane protein), an artificialhydrophobic sequence, or a combination thereof. While specific examplesare provided herein and used in the Examples, other transmembranedomains will be apparent to those of skill in the art and can be used inconnection with alternate embodiments of the technology. A selectedtransmembrane region or fragment thereof would preferably not interferewith the intended function of the CAR. As used in relation to atransmembrane domain of a protein or polypeptide, “fragment thereof”refers to a portion of a transmembrane domain that is sufficient toanchor or attach a protein to a cell surface.

In one embodiment, a CAR's transmembrane domain or fragment thereof isderived from or comprises the transmembrane domain of CD8. In analternate embodiment, the transmembrane domain or fragment thereof ofthe CAR described herein comprises a transmembrane domain selected fromthe transmembrane domain of an alpha, beta or zeta chain of a T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27,LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR,HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2Rgamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb,ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108),SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.

CD8 is an antigen preferentially found on the cell surface of cytotoxicT lymphocytes. CD8 mediates cell-cell interactions within the immunesystem, and acts as a T cell coreceptor. CD8 consists of an alpha (CD8a)and beta (CD8(3) chain. CD8a sequences are known for a number ofspecies, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBIRef Seq NP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8can refer to human CD8, including naturally occurring variants,molecules, and alleles thereof. In some embodiments of any of theaspects, e.g., in veterinary applications, CD8 can refer to the CD8 of,e.g., dog, cat, cow, horse, pig, and the like. Homologs and/or orthologsof human CD8 are readily identified for such species by one of skill inthe art, e.g., using the NCBI ortholog search function or searchingavailable sequence data for a given species for sequence similar to areference CD8 sequence.

Co-Stimulatory Domain

Each CAR described herein comprises an intracellular domain of aco-stimulatory molecule, or co-stimulatory domain. As used herein, theterm “co-stimulatory domain” refers to an intracellular signaling domainof a co-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD3θ,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, the intracellular domain is the intracellular domain of4-1BB.

4-1BBL is a type 2 transmembrane glycoprotein belonging to the TNFsuperfamily. 4-1BBL is expressed on activated T lymphocytes. 4-1BBLsequences are known for a number of species, e.g., human 4-1BBL, alsoknown as TNFSF9 (NCBI Gene ID: 8744) polypeptide (e.g., NCBI Ref SeqNP_003802.1) and mRNA (e.g., NCBI Ref Seq NM_003811.3). 4-1BBL can referto human 4-1BBL, including naturally occurring variants, molecules, andalleles thereof. In some embodiments of any of the aspects, e.g., inveterinary applications, 4-1BBL can refer to the 4-1BBL of, e.g., dog,cat, cow, horse, pig, and the like. Homologs and/or orthologs of human4-1BBL are readily identified for such species by one of skill in theart, e.g., using the NCBI ortholog search function or searchingavailable sequence data for a given species for sequence similar to areference 4-1BBL sequence.

Intracellular Signaling Domain

CARs as described herein comprise an intracellular signaling domain. An“intracellular signaling domain,” refers to the part of a CARpolypeptide that participates in transducing the message of effectiveCAR binding to a target antigen into the interior of the immune effectorcell to elicit effector cell function, e.g., activation, cytokineproduction, proliferation and cytotoxic activity, including the releaseof cytotoxic factors to the CAR-bound target cell, or other cellularresponses elicited following antigen binding to the extracellular CARdomain.

CD3 is a T cell co-receptor that facilitates T lymphocytes activationwhen simultaneously engaged with the appropriate co-stimulation (e.g.,binding of a co-stimulatory molecule). A CD3 complex consists of 4distinct chains; mammal CD3 consists of a CD3γ chain, a CD3δchain, andtwo CD3ε chains. These chains associate with a molecule known as the Tcell receptor (TCR) and the CD3ζ to generate an activation signal in Tlymphocytes. A complete TCR complex comprises a TCR, CD3ζ, and thecomplete CD3 complex.

In some embodiments of any aspect, a CAR polypeptide described hereincomprises an intracellular signaling domain that comprises anImmunoreceptor Tyrosine-based Activation Motif or ITAM from CD3 zeta(CD3ζ). In some embodiments of any aspect, the ITAM comprises threemotifs of ITAM of CD3ζ (ITAM3). In some embodiments of any aspect, thethree motifs of ITAM of CD3 are mutated.

ITAMs are known as a primary signaling domains which regulate primaryactivation of the TCR complex either in a stimulatory way, or in aninhibitory way. Primary signaling domains that act in a stimulatorymanner may contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Non-limiting examples ofITAM-containing intracellular signaling domains that are of particularuse in the technology include those derived from TCRζ, FcRγ, FcRβ, CD3γ,CD3θ, CD3ε, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

One skilled in the art will be capable of introducing mutations into thenucleic acid sequence of a gene or gene product, for example an ITAM,using standard techniques. For example, point mutations can beintroduced via site-directed point mutagenesis, a PCR technique.Site-directed mutagenesis kits are commercially available, for instance,through New England Biolabs; Ipswich, Mass. Non-limiting examples ofalternative methods to introduce point mutations to the nucleic acidsequence of a gene or gene product include cassette mutagenesis or wholeplasmid mutagenesis.

In one embodiment, the ITAM utilized in the CAR is based on alternativesto CD3ζ, including mutated ITAMs from CD3ζ (which contains 3 ITAMmotifs), truncations of CD3, and alternative splice variants known asCD3ε, CD3θ, and artificial constructs engineered to express fusionsbetween CD3ε or CD3θ and CD3ζ.

In one embodiment, the CD3ζ ITAM3 sequence corresponds to the sequenceof SEQ ID NO: 13.; or comprises the sequence of SEQ ID NO: 13; orcomprises a sequence with at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% orgreater sequence identity to the sequence of SEQ ID NO: 13.

In one embodiment, the intracellular signaling domain comprises a CD3ζITAM3 sequence selected from SEQ ID NOs: 13, 14, 33, or 34. In oneembodiment, the tyrosine residues are mutated to phenylalanine residues,thereby inhibiting the phosphorylation of the native tyrosine residues.Exemplary tyrosine residues that can be mutated include any tyrosineresidue described in Examples 9 or 10. It is contemplated that thetyrosine residues can be mutated to any residue that results in theinhibition of tyrosine phosphorylation. In another embodiment of anyaspect, tyrosines are mutated in at least one, at least two, or allthree ITAMs (e.g., ITAM I, II, and III).

In one embodiment, the T-cell intracellular signaling domain comprisesthe ITAMs of CD3 eta (CDR), CD3 theta (CD3θ) or CD3. In one embodiment,the T-cell intracellular signaling domain is the ITAM of CD3 eta (CDR),CD3 theta (CD3θ) or CD3.

In one embodiment, the intracellular signaling domain comprises an CD3ζITAM3 sequence comprising a deletion relative to the CD3ζ ITAM3 sequenceof SEQ ID NO: 13.

A deletion relative to the CD3ζ ITAM3 sequence can be performed usingtechniques well known in the art, for example, CRISPR, TALEN or ZFNtechnology. Methods of engineering nucleases to achieve a desiredsequence specificity are known in the art and are described, e.g., inKim (2014); Kim (2012); Belhaj et al. (2013); Urnov et al. (2010);Bogdanove et al. (2011); Jinek et al. (2012) Silva et al. (2011); Ran etal. (2013); Carlson et al. (2012); Guerts et al. (2009); Taksu et al.(2010); and Watanabe et al. (2012); each of which is incorporated byreference herein in its entirety. In alternate embodiments of anyaspect, a deletion relative to the ITAM3 sequence can be done viainhibitory nucleic acid. As used herein, “inhibitory nucleic acid”refers to a nucleic acid molecule which can inhibit the expression of atarget, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs),and the like. Inhibitory nucleic acid technology is more fully describedin Wilson, R C, and Doudna, J A. (2103) Annual Review of Biophysics42(217-239) and reference cited therein.

Experimental data provided herein show that variants of theintracellular signaling domain described herein results in a moreefficient CAR T cell. It is expected that replacing the intracellularsignaling domain of any CAR T cell with any intracellular signalingdomain variant described herein would improve the efficacy of the CAR Tcell.

A more detailed description of CARs and CAR T cells can be found in Mauset al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 201416:1441-1458; Hoyos et al. Haematologica 2012 97:1622; Byrd et al. JClin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562;and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which isincorporated by reference herein in its entirety.

In one embodiment, the CAR further comprises a linker domain. As usedherein “linker domain” refers to an oligo- or polypeptide region fromabout 2 to 100 amino acids in length, which links together any of thedomains/regions of the CAR as described herein. In some embodiment,linkers can include or be composed of flexible residues such as glycineand serine so that the adjacent protein domains are free to moverelative to one another. Longer linkers may be used when it is desirableto ensure that two adjacent domains do not sterically interfere with oneanother. Linkers may be cleavable or non-cleavable. Examples ofcleavable linkers include 2A linkers (for example T2A), 2A-like linkersor functional equivalents thereof and combinations thereof. In oneembodiment, the linker region is T2A derived from Thosea asigna virus.Non-limiting examples of linkers that can be used in this technologyinclude P2A and F2A.

In one embodiment, a CAR as described herein further comprises areporter molecule, e.g., to permit for non-invasive imaging (e.g.,positron-emission tomography PET scan). In a bispecific CAR thatincludes a reporter molecule, the first extracellular binding domain andthe second extracellular binding domain can include different or thesame reporter molecule. In a bispecific CAR T cell, the first CAR andthe second CAR can express different or the same reporter molecule. Inanother embodiment, a CAR as described herein further comprises areporter molecule (for example hygromycin phosphotransferase (hph)) thatcan be imaged alone or in combination with a substrate or chemical (forexample 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine ([¹⁸F]FHBG)). Inanother embodiment, a CAR as described herein further comprisesnanoparticles at can be readily imaged using non-invasive techniques(e.g., gold nanoparticles (GNP) functionalized with ⁶⁴Cu²⁺). Labeling ofCAR T cells for non-invasive imaging is reviewed, for example inBhatnagar P, et al. Integr Biol. (Camb). 2013 January; 5(1): 231-238,and Keu K V, et al. Sdci Transl Med. 2017 Jan. 18; 9(373), which areincorporated herein by reference in their entireties.

GFP and mCherry are demonstrated herein as fluorescent tags useful forimaging a CAR expressed on a T cell (e.g., a CAR T cell). It is expectedthat essentially any fluorescent protein known in the art can be used asa fluorescent tag for this purpose. For clinical applications, the CARneed not include a fluorescent tag or fluorescent protein.

In one embodiment, the CAR polypeptide comprises a BCMA binding domain,CD8 transmembrane domain, 4-1 BB costimulatory domain, a CD3ζ signalingdomain, a T2A linker, and an mCherry protein. In another embodiment, theCD3ζ domain can be replaced with CD3ε, CD3θ, and/or otherITAM-containing variants of CD3-related signaling domains.

In one embodiment, the CAR polypeptide sequence corresponds to, orcomprises, or comprises a sequence with at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% orgreater sequence identity of a sequence selected from SEQ ID NO: 15 or35-40.

In one embodiment, the CAR polypeptide comprises a CD37 binding domain,CD8 transmembrane domain, 4-1 BB co-stimulatory domain, a CD3ζ signalingdomain, a T2A linker, and an mCherry protein. In another embodiment, theCD3ζ domain can be replaced with CD3ε, CD3θ, and/or otherITAM-containing variants of CD3-related signaling domains.

In one embodiment, the CAR polypeptide sequence corresponds to, orcomprises, or comprises a sequence with at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% orgreater sequence identity of a sequence selected from SEQ ID NO: 21 or27.

One aspect of the technology described herein relates to a mammaliancell comprising any of the CAR polypeptides described herein; or anucleic acid encoding any of the CAR polypeptides described herein. Inone embodiment, the mammalian cell comprises an antibody, antibodyreagent, antigen-binding portion thereof, or any of the CARs describedherein, or a nucleic acid encoding such an antibody, antibody reagent,antigen-binding portion thereof, or any of the CARs described herein.The mammalian cell or tissue can be of human, primate, hamster, rabbit,rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any othermammalian cell may be used. In a preferred embodiment of any aspect, themammalian cell is human.

In one embodiment, the cell is a T cell. In alternate embodiments of anyaspect, the cell is an immune cell. As used herein, “immune cell” refersto a cell that plays a role in the immune response. Immune cells are ofhematopoietic origin, and include lymphocytes, such as B cells and Tcells; natural killer cells; myeloid cells, such as monocytes,macrophages, eosinophils, mast cells, basophils, and granulocytes. Insome embodiments, the cell is a T cell; a NK cell; a NKT cell;lymphocytes, such as B cells and T cells; and myeloid cells, such asmonocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes.

In one embodiment, the cell is obtained from an individual having ordiagnosed as having cancer, a plasma cell disorder, or autoimmunedisease.

“Cancer” as used herein can refer to a hyperproliferation of cells whoseunique trait— loss of normal cellular control—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis,and can be leukemia, lymphoma, multiple myeloma, or a solid tumor.Non-limiting examples of leukemia include acute myeloid leukemia (AML),Chronic myeloid leukemia (CML), Acute lymphocytic leukemia (ALL), andChronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALLor CLL. Non-limiting examples of lymphoma include Diffuse large B-celllymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia(CLL), Small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL),Marginal zone lymphomas, Burkitt lymphoma, hairy cell leukemia (HCL),and T cell lymphoma (e.g., peripheral T cell lymphoma (PTCL), includingcutaneous T cell lymphoma (CTCL) and anaplastic large cell lymphoma(ALCL)). In one embodiment, the cancer is DLBCL or Follicular lymphoma.Non-limiting examples of solid tumors include Adrenocortical Tumor,Alveolar Soft Part Sarcoma, Carcinoma, Chondrosarcoma, ColorectalCarcinoma, Desmoid Tumors, Desmoplastic Small Round Cell Tumor,Endocrine Tumors, Endodermal Sinus Tumor, EpithelioidHemangioendothelioma, Ewing Sarcoma, Germ Cell Tumors (Solid Tumor),Giant Cell Tumor of Bone and Soft Tissue, Hepatoblastoma, HepatocellularCarcinoma, Melanoma, Nephroma, Neuroblastoma, Non-Rhabdomyosarcoma SoftTissue Sarcoma (NRSTS), Osteosarcoma, Paraspinal Sarcoma, Renal CellCarcinoma, Retinoblastoma, Rhabdomyosarcoma, Synovial Sarcoma, and WilmsTumor. Solid tumors can be found in bones, muscles, or organs, and canbe sarcomas or carcinomas. It is contemplated that any aspect of thetechnology described herein can be used to treat all types of cancers,including cancers not listed in the instant application. As used herein,the term “tumor” refers to an abnormal growth of cells or tissues, e.g.,of malignant type or benign type.

As used herein, an “autoimmune disease or disorder” is characterized bythe inability of one's immune system to distinguish between a foreigncell and a healthy cell. This results in one's immune system targetingone's healthy cells for programmed cell death. Non-limiting examples ofan autoimmune disease or disorder include inflammatory arthritis, type 1diabetes mellitus, multiples sclerosis, psoriasis, inflammatory boweldiseases, SLE, and vasculitis, allergic inflammation, such as allergicasthma, atopic dermatitis, and contact hypersensitivity. Other examplesof auto-immune-related disease or disorder, but should not be construedto be limited to, include rheumatoid arthritis, multiple sclerosis (MS),systemic lupus erythematosus, Graves' disease (overactive thyroid),Hashimoto's thyroiditis (underactive thyroid), celiac disease, Crohn'sdisease and ulcerative colitis, Guillain-Barre syndrome, primary biliarysclerosis/cirrhosis, sclerosing cholangitis, autoimmune hepatitis,Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture'ssyndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporalarteritis/giant cell arteritis, chronic fatigue syndrome CFS),psoriasis, autoimmune Addison's Disease, ankylosing spondylitis, Acutedisseminated encephalomyelitis, antiphospholipid antibody syndrome,aplastic anemia, idiopathic thrombocytopenic purpura, Myasthenia gravis,opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis,pemphigus, pernicious anaemia, polyarthritis in dogs, Reiter's syndrome,Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener'sgranulomatosis and fibromyalgia (FM).

In one embodiment, the mammalian cell is obtained for a patient havingan immune system disorder that results in abnormally low activity of theimmune system, or immune deficiency disorders, which hinders one'sability to fight a foreign cell, (i.e., a virus or bacterial cell).

A plasma cell is a white blood cell produces from B lymphocytes whichfunction to generate and release antibodies needed to fight infections.As used herein, a “plasma cell disorder or disease” is characterized byabnormal multiplication of a plasma cell. Abnormal plasma cells arecapable of “crowding out” healthy plasma cells, which results in adecreased capacity to fight a foreign object, such as a virus orbacterial cell. Non-limiting examples of plasma cell disorders includeamyloidosis, Waldenstrom's macroglobulinemia, osteosclerotic myeloma(POEMS syndrome), Monoclonal gammopathy of unknown significance (MGUS),and plasma cell myeloma.

T cells can be obtained from a subject using standard techniques knownin the field. For example, T cells can be isolated from peripheral bloodtaken from a donor or patient. T cells can be isolated from a mammal.Preferably, T cells are isolated from a human.

A cell, for example a T cell, can be engineered to comprise any of theCAR polypeptides described herein; or a nucleic acid encoding any of theCAR polypeptides described herein. In one embodiment, the any of the CARpolypeptides described herein are comprised in a lentiviral vector. Thelentiviral vector is used to express the CAR polypeptide in a cell usinginfection standard techniques.

Retroviruses, such as lentiviruses, provide a convenient platform fordelivery of nucleic acid sequences encoding a gene, or chimeric gene ofinterest. A selected nucleic acid sequence can be inserted into a vectorand packaged in retroviral particles using techniques known in the art.The recombinant virus can then be isolated and delivered to cells, e.g.in vitro or ex vivo. Retroviral systems are well known in the art andare described in, for example, U.S. Pat. No. 5,219,740; Kurth andBannert (2010) “Retroviruses: Molecular Biology, Genomics andPathogenesis” Calster Academic Press (ISBN:978-1-90455-55-4); and Hu andPathak Pharmacological Reviews 2000 52:493-512; which are incorporatedby reference herein in their entirety. Lentiviral system for efficientDNA delivery can be purchased from OriGene; Rockville, Md. Inalternative embodiments, the CAR polypeptide of any of the CARSdescribed herein are expressed in the mammalian cell via transfection orelectroporation of an expression vector comprising nucleic acid encodingthe CAR. Transfection or electroporation methods are known in the art.

Efficient expression of the CAR polypeptide of any of the CARpolypeptides described herein can be assessed using standard assays thatdetect the mRNA, DNA, or gene product of the nucleic acid encoding theCAR. For example, RT-PCR, FACS, northern blotting, western blotting,ELISA, or immunohistochemistry.

In one embodiment, the CAR polypeptide described herein isconstitutively expressed. In one embodiment, the CAR polypeptidedescribed herein is encoded by recombinant nucleic acid sequence.

One aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: engineering a T cell tocomprise any of the CAR polypeptides described herein on the T cellsurface; and administering the engineered T cell to the subject.

One aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: administering the cellof any of the mammalian cells comprising the any of the CAR polypeptidesdescribed herein.

Cluster differentiation (CD) molecules are cell surface markers presenton leukocytes. As a leukocyte differentiates and matures its CD profilechanges. In the case that a leukocytes turns into a cancer cell, (i.e.,a lymphoma), its CD profile is important in diagnosing the disease. Thetreatment and prognosis of certain types of cancers is reliant ondetermining the CD profile of the cancer cell. “CDX+”, wherein “X” is aCD marker, indicates the CD marker is present in the cancer cell, while“CDX−” indicates the marker is not present. One skilled in the art willbe capable of assessing the CD molecules present on a cancer cell usingstandard techniques, for example using immunofluorescence to detectcommercially available antibodies bound to the CD molecules.

In one embodiment, the cancer expresses a CD and tumor antigen. In oneembodiment, the cancer is a CD37+ or BCMA+ cancer. In one embodiment,the CD37+ cancer is lymphoma or leukemia. In one embodiment, lymphoma isB-cell Non-Hodgkin Lymphoma (NHL), mantle cell lymphoma, Burkitt'slymphoma, B cell lymphoblastic lymphoma or T cell lymphoma (e.g.,peripheral T cell lymphoma (PTCL), including cutaneous T-cell lymphoma(CTCL) and anaplastic large cell lymphoma (ALCL)).

One aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: engineering a T cell tocomprise any of the CAR polypeptides described herein on the T cellsurface; administering the engineered T cell to the subject; wherein thesubject is non-responsive to anti-CD19 and/or anti-CD20 therapy.

Cancer cells can evolve in response to treatment to alter its CD profilein order to evade said treatment. For example, a patient with CD19+leukemia can be treated with an anti-CD19 therapy. Following treatment,the cancer cell can relapse, or come back after treatment, and no longerexpress the CD19 marker, resulting in CD19− leukemia. This cancer cellwill no longer be targetable by an anti-CD19 therapy. In one embodiment,the subject is non-responsive, or refractory to anti-CD19 and/oranti-CD20 therapy. In one embodiment, the subject has a cancer that isCD19− and/or CD20−. In one embodiment, the subject has a cancer that isrelapsed and no longer expresses CD19 or CD20.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising administering any of themammalian cells comprising the any of the CAR polypeptides describedherein to the subject, wherein the cell comprises CAR comprising anextracellular domain comprising a CD37-binding sequence; wherein thesubject is non-responsive to anti-CD19 and/or anti-CD20 therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: selecting a subject whois non-responsive to anti-CD19 and/or anti-CD20 therapy; engineering a Tcell to comprise any of the CAR polypeptides described herein on the Tcell surface; administering the engineered CAR T cell to the subject;wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: selecting a subject whois non-responsive to anti-CD19 and/or anti-CD20 therapy; administeringany of the mammalian cells comprising the any of the CAR polypeptidesdescribed herein to the subject, wherein the cell comprises CARcomprising an extracellular domain comprising a CD37-binding sequence;wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising: engineering a T cell tocomprise any of the CAR polypeptides described herein on the T cellsurface; administering the engineered CAR T cell to the subject; whereinthe subject is concurrently administered an anti-CD19 and/or anti-CD20therapy.

Another aspect of the technology described herein relates to a method oftreating cancer, a plasma cell disorder, or an autoimmune disease in asubject in need thereof, the method comprising administering any of themammalian cells comprising the any of the CAR polypeptides describedherein to the subject, wherein the cell comprises CAR comprising anextracellular domain comprising a CD37-binding sequence; wherein thesubject is concurrently administered an anti-CD19 and/or anti-CD20therapy.

In some embodiments of any of the aspect, the engineered CAR-T cell isstimulated and/or activated prior to administration to the subject.

Administration

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having cancer, a plasma cell disease ordisorder, or an autoimmune disease or disorder with a mammalian cellcomprising any of the CAR polypeptides described herein, or a nucleicacid encoding any of the CAR polypeptides described herein. As usedherein, a “CAR T cells as described herein” refers to a mammalian cellcomprising any of the CAR polypeptides described herein, or a nucleicacid encoding any of the CAR polypeptides described herein. As usedherein, a “condition” refers to a cancer, a plasma cell disease ordisorder, or an autoimmune disease or disorder. Subjects having acondition can be identified by a physician using current methods ofdiagnosing the condition. Symptoms and/or complications of thecondition, which characterize these conditions and aid in diagnosis arewell known in the art and include but are not limited to, fatigue,persistent infections, and persistent bleeding. Tests that may aid in adiagnosis of, e.g. the condition, but are not limited to, bloodscreening and bone marrow testing, and are known in the art for a givencondition. A family history for a condition, or exposure to risk factorsfor a condition can also aid in determining if a subject is likely tohave the condition or in making a diagnosis of the condition.

The compositions described herein can be administered to a subjecthaving or diagnosed as having a condition. In some embodiments, themethods described herein comprise administering an effective amount ofactivated CAR T cells described herein to a subject in order toalleviate a symptom of the condition. As used herein, “alleviating asymptom of the condition” is ameliorating any condition or symptomassociated with the condition. As compared with an equivalent untreatedcontrol, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%,90%, 95%, 99% or more as measured by any standard technique. A varietyof means for administering the compositions described herein to subjectsare known to those of skill in the art. In one embodiment, thecompositions described herein are administered systemically or locally.In a preferred embodiment, the compositions described herein areadministered intravenously. In another embodiment, the compositionsdescribed herein are administered at the site of a tumor.

The term “effective amount” as used herein refers to the amount ofactivated CAR T cells needed to alleviate at least one or more symptomof the disease or disorder, and relates to a sufficient amount of thecell preparation or composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount ofactivated CAR T cells that is sufficient to provide a particularanti-condition effect when administered to a typical subject. Aneffective amount as used herein, in various contexts, would also includean amount sufficient to delay the development of a symptom of thedisease, alter the course of a symptom disease (for example but notlimited to, slowing the progression of a condition), or reverse asymptom of the condition. Thus, it is not generally practicable tospecify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be evaluatedby standard pharmaceutical procedures in cell cultures or experimentalanimals. The dosage can vary depending upon the dosage form employed andthe route of administration utilized. The dose ratio between toxic andtherapeutic effects is the therapeutic index and can be expressed as theratio LD50/ED50. Compositions and methods that exhibit large therapeuticindices are preferred. A therapeutically effective dose can be estimatedinitially from cell culture assays. Also, a dose can be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 (i.e., the concentration of activated CAR T cells,which achieves a half-maximal inhibition of symptoms) as determined incell culture, or in an appropriate animal model. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay, e.g., assay for bone marrow testing, among others. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment.

In one aspect of the technology, the technology described herein relatesto a pharmaceutical composition comprising activated CAR T cells asdescribed herein, and optionally a pharmaceutically acceptable carrier.The active ingredients of the pharmaceutical composition at a minimumcomprise activated CAR T cells as described herein. In some embodiments,the active ingredients of the pharmaceutical composition consistessentially of activated CAR T cells as described herein. In someembodiments, the active ingredients of the pharmaceutical compositionconsist of activated CAR T cells as described herein. Pharmaceuticallyacceptable carriers for cell-based therapeutic formulation includesaline and aqueous buffer solutions, Ringer's solution, and serumcomponent, such as serum albumin, HDL and LDL. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

In some embodiments, the pharmaceutical composition comprising activatedCAR T cells as described herein can be a parenteral dose form. Sinceadministration of parenteral dosage forms typically bypasses thepatient's natural defenses against contaminants, the components apartfrom the CAR T cells themselves are preferably sterile or capable ofbeing sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. Any of these can be added to the activated CART cells preparation prior to administration.

Suitable vehicles that can be used to provide parenteral dosage forms ofactivated CAR T cells as disclosed within are well known to thoseskilled in the art. Examples include, without limitation: salinesolution; glucose solution; aqueous vehicles including but not limitedto, sodium chloride injection, Ringer's injection, dextrose Injection,dextrose and sodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Dosage

“Unit dosage form” as the term is used herein refers to a dosage forsuitable one administration. By way of example a unit dosage form can bean amount of therapeutic disposed in a delivery device, e.g., a syringeor intravenous drip bag. In one embodiment, a unit dosage form isadministered in a single administration. In another, embodiment morethan one unit dosage form can be administered simultaneously.

In some embodiments, the activated CAR T cells described herein areadministered as a monotherapy, i.e., another treatment for the conditionis not concurrently administered to the subject.

A pharmaceutical composition comprising the T cells described herein cangenerally be administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, in some instances 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. If necessary, T cell compositionscan also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

In certain aspects, it may be desired to administer activated CAR Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom as described herein,and reinfuse the patient with these activated and expanded T cells. Thisprocess can be carried out multiple times every few weeks. In certainaspects, T cells can be activated from blood draws of from 10 cc to 400cc. In certain aspects, T cells are activated from blood draws of 20 cc,30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

Modes of administration can include, for example intravenous (i.v.)injection or infusion. The compositions described herein can beadministered to a patient transarterially, intratumorally, intranodally,or intramedullary. In some embodiments, the compositions of T cells maybe injected directly into a tumor, lymph node, or site of infection. Inone embodiment, the compositions described herein are administered intoa body cavity or body fluid (e.g., ascites, pleural fluid, peritonealfluid, or cerebrospinal fluid).

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates can be expanded by contact with an aAPC as described herein,e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs as described hereinand treated such that one or more CAR constructs of the technology maybe introduced, thereby creating a CAR T cell. Subjects in need thereofcan subsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. Following orconcurrent with the transplant, subjects can receive an infusion of theexpanded CAR T cells. In one embodiment, expanded cells are administeredbefore or following surgery.

In some embodiments, lymphodepletion is performed on a subject prior toadministering one or more CAR T cell as described herein. In suchembodiments, the lymphodepletion can comprise administering one or moreof melphalan, Cytoxan®, cyclophosphamide, and fludarabine.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.

In some embodiments, a single treatment regimen is required. In others,administration of one or more subsequent doses or treatment regimens canbe performed. For example, after treatment biweekly for three months,treatment can be repeated once per month, for six months or a year orlonger. In some embodiments, no additional treatments are administeredfollowing the initial treatment.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to administer further cells, discontinue treatment, resumetreatment, or make other alterations to the treatment regimen. Thedosage should not be so large as to cause adverse side effects, such ascytokine release syndrome. Generally, the dosage will vary with the age,condition, and sex of the patient and can be determined by one of skillin the art. The dosage can also be adjusted by the individual physicianin the event of any complication.

Combinational Therapy

The activated CAR T cells described herein can be used in combinationwith other known agents and therapies. In one embodiment, the subject isfurther administered an anti-BCMA therapy. In one embodiment, thesubject is resistant to anti-BCMA therapies. Administered “incombination,” as used herein, means that two (or more) differenttreatments are delivered to the subject during the course of thesubject's affliction with the disorder, e.g., the two or more treatmentsare delivered after the subject has been diagnosed with the disorder andbefore the disorder has been cured or eliminated or treatment has ceasedfor other reasons. In some embodiments, the delivery of one treatment isstill occurring when the delivery of the second begins, so that there isoverlap in terms of administration. This is sometimes referred to hereinas “simultaneous” or “concurrent delivery.” In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered. The activated CAR T cells described herein and the at leastone additional therapeutic agent can be administered simultaneously, inthe same or in separate compositions, or sequentially. For sequentialadministration, the CAR-expressing cell described herein can beadministered first, and the additional agent can be administered second,or the order of administration can be reversed. The CAR T therapy and/orother therapeutic agents, procedures or modalities can be administeredduring periods of active disorder, or during a period of remission orless active disease. The CAR T therapy can be administered beforeanother treatment, concurrently with the treatment, post-treatment, orduring remission of the disorder.

When administered in combination, the activated CAR T cells and theadditional agent (e.g., second or third agent), or all, can beadministered in an amount or dose that is higher, lower or the same asthe amount or dosage of each agent used individually, e.g., as amonotherapy. In certain embodiments, the administered amount or dosageof the activated CAR T cells, the additional agent (e.g., second orthird agent), or all, is lower (e.g., at least 20%, at least 30%, atleast 40%, or at least 50%) than the amount or dosage of each agent usedindividually. In other embodiments, the amount or dosage of theactivated CAR T cells, the additional agent (e.g., second or thirdagent), or all, that results in a desired effect (e.g., treatment ofcancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or atleast 50% lower) than the amount or dosage of each agent individuallyrequired to achieve the same therapeutic effect. In further embodiments,the activated CAR T cells described herein can be used in a treatmentregimen in combination with surgery, chemotherapy, radiation, an mTORpathway inhibitor, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH®, anti-CD3 antibodies orother antibody therapies, Cytoxan®, fludarabine, rapamycin, mycophenolicacid, steroids, FR901228, cytokines, or a peptide vaccine, such as thatdescribed in Izumoto et al. 2008 J Neurosurg 108:963-971.

In one embodiment, the activated CAR T cells described herein can beused in combination with a checkpoint inhibitor. Exemplary checkpointinhibitors include anti-PD-1 inhibitors (Nivolumab, MK-3475,Pembrolizumab, Pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors(Ipilimumab and Tremelimumab), anti-PDL1 inhibitors (Atezolizumab,Avelomab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3inhibitors.

In one embodiment, the activated CAR T cells described herein can beused in combination with a chemotherapeutic agent. Exemplarychemotherapeutic agents include an anthracycline (e.g., doxorubicin(e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine,vincristine, vindesine, vinorelbine), an alkylating agent (e.g.,cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), animmune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab,tositumomab), an antimetabolite (including, e.g., folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide). General chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan®), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®). Exemplary alkylating agents include, without limitation,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®,Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®,Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®,Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide(Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™) ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treanda®). Exemplary mTOR inhibitorsinclude, e.g., temsirolimus; ridaforolimus (formally known asdeferolimus, (1R,2R,45)-4-[(2R)-2[(1R,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04′9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RADOO1);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055);2-Amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine-(SEQ ID NO: 41), inner salt (SF1126, CAS 936487-67-1), and XL765.Exemplary immunomodulators include, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics). Exemplary anthracyclinesinclude, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin(lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, andrubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal(daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD,Novantrone®); epirubicin (Ellence^(TM)); idarubicin (Idamycin®, IdamycinPFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin;and desacetylravidomycin. Exemplary vinca alkaloids include, e.g.,vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine(Eldisine®)); vinblastine (also known as vinblastine sulfate,vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine(Navelbine®). Exemplary proteosome inhibitors include bortezomib(Velcade®); carfilzomib (PX-171-007,(5)-4-Methyl-N-((5)-1-(((5)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((5)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPT0052); ixazomib citrate(MLN-9708); delanzomib (CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(11S′)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912).

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In an embodiment, activated CAR T cells described herein areadministered to a subject in combination with a molecule that decreasesthe level and/or activity of a molecule targeting GITR and/or modulatingGITR functions, a molecule that decreases the Treg cell population, anmTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptortyrosine kinase inhibitor, a CDK4 inhibitor, and/or a BTK inhibitor.

Efficacy

The efficacy of activated CAR T cells in, e.g. the treatment of acondition described herein, or to induce a response as described herein(e.g. a reduction in cancer cells) can be determined by the skilledclinician. However, a treatment is considered “effective treatment,” asthe term is used herein, if one or more of the signs or symptoms of acondition described herein is altered in a beneficial manner, otherclinically accepted symptoms are improved, or even ameliorated, or adesired response is induced e.g., by at least 10% following treatmentaccording to the methods described herein. Efficacy can be assessed, forexample, by measuring a marker, indicator, symptom, and/or the incidenceof a condition treated according to the methods described herein or anyother measurable parameter appropriate. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% or more.

Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein.

Treatment includes any treatment of a disease in an individual or ananimal (some non-limiting examples include a human or an animal) andincludes: (1) inhibiting the disease, e.g., preventing a worsening ofsymptoms (e.g. pain or inflammation); or (2) relieving the severity ofthe disease, e.g., causing regression of symptoms. An effective amountfor the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy of a given approach can be assessed in animalmodels of a condition described herein, for example treatment of ALL.When using an experimental animal model, efficacy of treatment isevidenced when a statistically significant change in a marker isobserved.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priortechnology or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A chimeric antigen receptor (CAR) polypeptide comprising:

a. an extracellular domain comprising a target-binding sequence;

b. a transmembrane domain;

c. a co-stimulatory domain; and

d. a T cell intracellular signaling domain that lacks a functional ITAM3sequence.

-   2. The CAR polypeptide of paragraph 1, wherein the transmembrane    domain is the transmembrane domain from is CD8 or 4-1BB.-   3. The CAR polypeptide of paragraph 1, wherein the co-stimulatory    domain is the co-stimulatory domain of 4-1BB.-   4. The CAR polypeptide of any of paragraphs 1-3, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    selected from SEQ ID NOs: 13, 14, 33, or 34.-   5. The CAR polypeptide of any of paragraphs 1-4, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    comprising a deletion relative to the CD3ζ ITAM3 sequence of SEQ ID    NO: 13.-   6. The CAR polypeptide of any of paragraphs 1-5, wherein the T-cell    intracellular signaling domain is the intracellular signaling domain    of CD3ε or CD3θ.-   7. The CAR polypeptide of any of paragraphs 1-6, wherein the    target-binding sequence comprises a ligand of the target or an    antibody reagent that specifically binds the target.-   8. The CAR polypeptide of any of paragraphs 1-7, wherein the target    is B cell maturation antigen (BCMA).-   9. The CAR polypeptide of any of paragraphs 1-8, wherein the target    is CD37.-   10. The CAR polypeptide of any of paragraphs 1-9, wherein the    antibody reagent comprises an scFv.-   11. The CAR polypeptide of any of paragraphs 1-10, wherein the    antibody reagent has the sequence selected from SEQ ID NO: 1, 5, or    9.-   12. A chimeric antigen receptor (CAR) polypeptide comprising:

a. an extracellular domain comprising a BCMA-binding sequence;

b. a transmembrane domain of CD8;

c. a co-stimulatory domain of a 4-1BB; and

d. a T cell intracellular signaling domain that lacks a functional ITAM3sequence.

-   13. The CAR polypeptide of paragraph 12, wherein the BCMA-binding    sequence comprises a ligand of BCMA or an antibody reagent that    specifically binds BCMA.-   14. The CAR polypeptide of any of paragraphs 12-13, wherein the    antibody reagent comprises a scFv.-   15. The CAR polypeptide of any of paragraphs 12-14, wherein the    antibody reagent the sequence of SEQ ID NO: 1-   16. The CAR polypeptide of any of paragraphs 12-15, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    selected from SEQ ID NOs: 13, 14, 33, or 34.-   17. The CAR polypeptide of any of paragraphs 12-16, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    comprising a deletion relative to the CD3ζ ITAM3 sequence of SEQ ID    NO: 13.-   18. A chimeric antigen receptor (CAR) polypeptide comprising:

a. an extracellular domain comprising a CD37-binding sequence;

b. a transmembrane domain of CD8;

c. a co-stimulatory domain of a 4-1BB; and

d. a T cell intracellular signaling domain that lacks a functional ITAM3sequence.

-   19. The CAR polypeptide of paragraph 18, wherein the CD37-binding    sequence comprises a ligand of CD37 or an antibody reagent that    specifically binds BCMA.-   20. The CAR polypeptide of any of paragraphs 18-19, wherein the    antibody reagent comprises a scFv.-   21. The CAR polypeptide of any of paragraphs 18-20, wherein the scFv    has a sequence selected from SEQ ID NO: 5 or 9.-   22. The CAR polypeptide of any of paragraphs 18-21, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    selected from SEQ ID NOs: 13, 14, 33, or 34.-   23. The CAR polypeptide of any of paragraphs 18-22, wherein the    intracellular signaling domain comprises a CD3ζ ITAM3 sequence    comprising a deletion relative to the CD3ζ ITAM3 sequence of SEQ ID    NO: 13.-   24. A mammalian cell comprising:

a. the CAR polypeptide of any of paragraphs 1-23; or

b. a nucleic acid encoding any of the CAR polypeptides of any ofparagraphs 1-23.

-   25. The cell of paragraph 24, wherein the cell is a T cell.-   26. The cell of any of paragraphs 24-25, wherein the cell is a human    cell.-   27. The cell of any of paragraphs 24-26, wherein the cell is    obtained from an individual having or diagnosed as having cancer, a    plasma cell disorder, or autoimmune disease.-   28. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. engineering a T cell to comprise a CAR polypeptide of any ofparagraphs 1-22 on the T cell surface;

b. administering the engineered T cell to the subject.

-   29. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. administering the cell of any of paragraphs 24-28 to the subject.

-   30. The method of any of paragraphs 28-29, wherein the cancer is a    CD37+ or BCMA+ cancer.-   31. The method of any of paragraphs 28-30, wherein the CD37+ cancer    is lymphoma or leukemia.-   32. The method of paragraph 31, wherein the lymphoma is B-cell    Non-Hodgkin Lymphoma (NHL), mantle cell lymphoma, Burkitt's    lymphoma, B cell lymphoblastic lymphoma or T cell lymphoma (e.g.,    peripheral T cell lymphoma (PTCL), including cutaneous T-cell    lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)).-   33. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. engineering a T cell to comprise a CAR polypeptide of paragraph 18 onthe T cell surface;

b. administering the engineered T cell to the subject;

wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

-   34. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising administering a cell of any of paragraphs 24-27 to the    subject, wherein the cell comprises CAR comprising an extracellular    domain comprising a CD37-binding sequence;

wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

-   35. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. selecting a subject who is non-responsive to anti-CD19 and/oranti-CD20 therapy;

b. engineering a T cell to comprise a CAR polypeptide of paragraph 18 onthe T cell surface;

c. administering the engineered T cell to the subject;

wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

-   36. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. selecting a subject who is non-responsive to anti-CD19 and/oranti-CD20 therapy;

b. administering a cell of any of paragraphs 24-27 to the subject,wherein the cell comprises CAR comprising an extracellular domaincomprising a CD37-binding sequence;

wherein the subject is non-responsive to anti-CD19 and/or anti-CD20therapy.

-   37. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising:

a. engineering a T cell to comprise a CAR polypeptide of paragraph 18 onthe T cell surface;

b. administering the engineered T cell to the subject;

wherein the subject is concurrently administered an anti-CD19 and/oranti-CD20 therapy.

-   38. A method of treating cancer, a plasma cell disorder, or an    autoimmune disease in a subject in need thereof, the method    comprising administering a cell of any of paragraphs 24-27 to the    subject, wherein the cell comprises CAR comprising an extracellular    domain comprising a CD37-binding sequence;

wherein the subject is concurrently administered an anti-CD19 and/oranti-CD20 therapy.

-   39. A composition comprising the CAR T cells of paragraphs 1-27    formulated for the treatment of cancer.-   40. The composition of paragraph 39, further comprising a    pharmaceutically acceptable carrier.-   41. The method of paragraph 31, wherein the leukemia is acute    myeloid leukemia (AML).

EXAMPLES Example 1

Described herein are methods and compositions relating to “alternativesignal 1” domains based on alternatives to CD3 zeta, including mutatedITAMs from CD3ζ (which contains 3 ITAM motifs), truncations of CD3 zeta,and alternative splice variants known as CD3 eta, CD3θ, and artificialconstructs engineered to express fusions between CD3 eta or theta andzeta. Thus, described herein are alternative signal 1 transducers toform optimally signaling CARs. The resulting alternative CARs decouplethe cytotoxic signal from the activation-induced cell death signal whichis mediated by engagement of the CAR and activation of CD3 zeta, thusresulting in more potent CAR-alternative T cells compared toCAR-original T cells. These CAR T cells are contemplated for use in thetreatment of cancer, infectious diseases, and transplantationindications.

Example 2

Development and optimization of a genetically-engineered T cell therapyfor advanced multiple myeloma.

It is contemplated herein that T cells can be genetically engineered totarget the B cell maturation antigen (BCMA), a transmembrane receptorconstitutively expressed on multiple myeloma cells, for use as adoptiveimmunotherapy for advanced multiple myeloma.

The use of T cells engineered with a chimeric antigen receptor (CAR) inthe clinic has achieved unprecedented results in the treatment ofleukemias and lymphomas in the past five years. CAR T cells targetingCD19, a molecule expressed on leukemic B cells, induce completeremission in—95% of patients suffering from relapsed or refractory (r/r)acute lymphoblastic leukemia (ALL). The remarkable effects of this noveltherapy have also been observed in patients with (r/r) follicularlymphoma, diffuse large B cell lymphoma, and chronic lymphocyticleukemia (3,5,6,9).

B-cell maturation antigen (BCMA) is a member of the tumor necrosisreceptor family (TNRF) which is constitutively expressed as atransmembrane protein in terminally differentiated B cells. Binding ofBCMA with its cognate ligands results in the generation of pro-survivaland proliferative cues to mature B cells, plasma cells, and multiplemyeloma cells (10). Described herein is the development of a CAR basedon the single-chain variable region (scFv) of a monoclonal antibody(mAb) specific for human BCMA(11). In addition, multiple versions ofthis CAR are examined for decoupling of T-cell intrinsic functions suchas cellular expansion and cytokine production. The approach examinesmodification of signaling capacity of CAR T cells to dampen cytokinerelease without affecting their proliferative potential, as the majorCAR T-cell-associated toxicity has been associated with the productionof cytokines (clinically defined as cytokine release syndrome, CRS).Finally, these modified CARs can be utilized in pre-clinical studies andin a dose-escalating phase I clinical trial to treat patients sufferingfrom advanced multiple myeloma. The generic configuration of theproposed BCMA-targeted CAR is shown in FIG. 1 .

Described herein is the characterization and validation of aBCMA-targeted chimeric antigenic receptor (CAR) to treat advancedmultiple myeloma, e.g., a BCMA-specific CAR is described herein.Preliminary data indicate that primary human T cells transduced withthis CAR (FIG. 1 ) have anti-myeloma cytotoxic activity in vitro (FIGS.2A-2C). The production of anti-BCMA CAR T cells can be optimized bymodifying the components of the CAR (scFv and intracellular domains).Established reagents are available for the detection of this CAR by flowcytometry and qPCR. This CAR can be further characterized using in vitrofunctional analyses such as cytokine production, proliferation, tumorlysis, and transduction efficiency assays. In addition, multipleBCMA-targeted CARs with modifications in the CD3ζ signaling domain.Specifically, variants of this CAR can be produced by mutating tyrosineto phenylalanine as single-point mutations in all three activationmodules, or ITAMs (I, II, and III), within the CD3ζ domain (shown inFIG. 1 ). These ITAMs are phosphorylated upon T cell activation and havebeen associated with proliferation, cytokine secretion, cytotoxicity,and activation-induced cell death (8). Described herein is theexamination of the functional capacity and the persistence of these CARmutants in comparison with the wild-type version. Alterations within theTCR signaling domain result in the dissociation of T-cell effectorfunctions (i.e., cytokine production, cytotoxicity) and proliferation.This has been shown to occur in mouse T cells (Rosenberg), but has notpreviously been demonstrated in human T cells nor in the context of aCAR molecule. Described herein is the transduction of normal human Tcells with various CAR configurations and testing of them againstmyeloma cell lines such as RPMI-8226 and U266, and against primary humanmyeloma tumor cells obtained from patients at MGH under an IRB-approvedprotocol. The responsiveness of the CAR T cells is also tested againstcell lines that express varying degrees of BCMA (FIG. 2A).

To validate BCMA-directed CAR T cells using xenogeneic models ofmultiple myeloma. Immunodeficient NSG mice grafted with human tumors andhuman T cells have emerged as the standard pre-clinical animal model inwhich to test CAR T cell functions. A xenograft model of multiplemyeloma (FIG. 3 ) was developed in the context of a different CAR (whichis not suitable for clinical translation but can serve as a control).Briefly, luciferase-expressing myeloma cells are injected in NSG mice atday 0. BCMA-directed CAR T cells are injected intravenously at day 6(allowing some time for tumor engraftment) at different concentrations(1, 5, and 10 million CAR T cells/mouse) along with appropriate cohortsof mice injected with untransduced T cell controls. Mice are imagedweekly to determine tumor burden by bioluminescence. This assay can beused to develop a CAR T cell with the most optimal tumor-killing,proliferative, and survival capabilities.

Statistical Analysis

Since most of these experiments are performed simultaneously incomparison to a single control dataset, the majority of the results areanalyzed by unpaired Student's t-tests. p-values less than 0.05 asstatistically significant. All experiments will be repeated with T cellsfrom at least 3 healthy donors.

REFERENCES

-   1. Melenhorst J J, Lacey S., Bedoya F. Chimeric antigen receptor T    cells: Self-replicating drugs for cancer. Curr Drug Targets (2015).-   2. Maus, M. V., Grupp, S. A., Porter, D. L. & June, C. H. Antibody    modified T cells: CARs take the front seat for hematologic    malignancies. Blood (2014).-   3. Maus, M. V., et al. Adoptive immunotherapy for cancer or viruses.    Annual review of immunology 32, 189-225 (2014).-   4. Sadelain, M., Riviere, I. & Brentjens, R. Targeting tumours with    genetically enhanced T lymphocytes. Nature reviews. Cancer 3, 35-45    (2003).-   5. Grupp, S. A., et al. Chimeric antigen receptor-modified T cells    for acute lymphoid leukemia. New England Journal of Medicine 368,    1509-1518 (2013).-   6. Maude, S. L., Teachey, D. T., Porter, D. L. & Grupp, S. A.    CD19-targeted chimeric antigen receptor T-cell therapy for acute    lymphoblastic leukemia. Blood 125, 4017-4023 (2015).-   7. Ledford, H. Immunotherapy's cancer remit widens. Nature 497, 544    (2013). Combadiere, B., et al. Qualitative and quantitative    contributions of the T cell receptor zeta chain to mature T cell    apoptosis. The Journal of experimental medicine 183, 2109-2117    (1996).-   8. Kochenderfer, J. N., Yu, Z., Frasheri, D., Restifo, N. P. &    Rosenberg, S. A. Adoptive transfer of syngeneic T cells transduced    with a chimeric antigen receptor that recognizes murine CD19 can    eradicate lymphoma and normal B cells. Blood 116, 3875-3886 (2010).-   9, Carpenter, R. O., et al. B-cell maturation antigen is a promising    target for adoptive T-cell therapy of multiple myeloma. Clinical    cancer research: an official journal of the American Association for    Cancer Research 19, 2048-2060 (2013).-   10. Tai, Y. T., et al. Novel anti-B-cell maturation antigen    antibody-drug conjugate (GSK2857916) selectively induces killing of    multiple myeloma. Blood 123, 3128-3138 (2014).

Example 3

scFv from a humanized anti-BCMA mAb was used to construct anti-BCMA CARconstruct cloned into a lentivirus vector (FIG. 4 ). Intracellulardomains are 4-1BB (CD137) ICD linked to CD3ζ. ITAMS (I, II, and III)shown. T2A self-cleaving peptide followed by an mCherry reporter gene.

CARs with alternative intracellular signaling domains were constructed(FIG. 5 ) and tested for activation (FIG. 6 ). All CARs displayedequivalent cytotoxicity, except for BB-STOP CAR (FIG. 7 ). CAR-specificcytotoxicity was also tested over a range of concentrations (FIG. 8 ).

CAR-specific cytokine production was examined and no statisticallysignificant difference was found using 3 normal donors and one-way ANOVA(FIG. 9 ). CAR-specific proliferation was examined using 1 normal donorand no statistically significant differences were found between theCAR-Ts (FIG. 10 ).

Expression of the early activation marker CD69 was examined after CARstimulation (FIG. 11 ). No statistically significant difference wasfound between the CAR Ts.

CD107a degranulation was tested. ND29 degranulation was measured 5 hrafter CAR stimulation with K562-BCMA⁺ cells (FIG. 12 ). No statisticallysignificant difference was found between the CAR-Ts.

CD3ζ KO T cells were constructed and stained for CD3epsilon (also,CD3zeta, TCR alpha and beta). The cells were stimulated with K562-BCMA+cells (FIG. 13 ). The results show that there are no significantdifferences in the activation levels produced by the different CARstested. Accordingly, wild-type CD3ζ is not necessary for CARfunction—other alternatives are available (mutants, isoforms).

In in vivo assays, anti-BCMA CAR T Cells with mutITAM3 have betterkilling capacity and persistence (FIG. 14 ). Analysis of peripheralblood shows that CAR T cells that lack a functional ITAM3 have bettersurvival capacity that cells having an intact CD3ζ (3 ITAMs) at 64 daypost-infusion (FIG. 15 ).

CAR-T lacking ITAM3 were analyzed and found to differentiate into cellsassociated with better survival capacity such as naïve-like (stem cellmemory) and central memory T cells (FIG. 16 ).

Described herein are CAR T cells targeting BCMA that can efficientlylyse multiple myeloma cells. All the BCMA-CARs tested (CD3ζ mutants andisoforms) have similar tumoricidal capacity as CAR T cells bearing awild-type C CD3ζ domain. CARs with an inactive ITAM3 have betterpersistence in vivo, possibly due to higher percentage of stem cellmemory T cells (Tscm).

Example 4

A number of alternative CARs were designed (FIG. 20 ). Activation andcytotoxicity of the resulting CAR Ts were tested (FIGS. 21A & 21B) butno significant difference in activation and in-vitro cytotoxicity of CART cells with signaling-deficient ITAM1 were detected.

To determine if the CD3ζ domain was necessary for CAR function,4-1BBSTOP CAR was constructed (FIG. 22 ). The transduction efficiency ofthe CAR constructs was tested with Jurkat T cells (FIG. 24 ) and primaryT cells (FIG. 26 ). Activation assays with anti-BCMA Jurkat T cells areshown in FIG. 25 .

The tested constructs demonstrated tumor killing activity (FIG. 27 ) andproliferation in different T cell lines (FIGS. 28 and 29 ).

In vivo cytotoxicity was tested using 20 NSG mice (4 mice/condition)(FIG. 30 ). All mice were injected with 1e6 U266 cells i.v. on day 0.Anti-BCMA ND23 CAR T cells (1e6/mouse) and UTDs were injected at day 7.The CAR T were: UTD; WT; MutITAM3; MutITAM1+3; and BB-Stop. Mice wereimaged every week to analyze tumor burden (FIG. 31 ; FIG. 32 ). At day64, the mice were bled and analyzed for levels of anti-BCMA positive/CD3positive cells (FIG. 33 ).

Example 5

Described herein is the use of genetically modified T cells expressinganti-CD37 chimeric antigen receptor (CAR) to treat CD37 positivemalignancies including B cell-NHL. The CAR comprises CD37 bindingdomain, CD8 transmembrane domain, 4-1 BB costimulatory signaling region,and a CD3ζ signaling domain. In another embodiment, the CD3ζ domain canbe replaced with CD3 eta, CD3 theta, and/or other ITAM-containingvariants of CD3-related signaling domains. CAR T cells are generated byintroducing a lentiviral vector comprising CD37-CAR in primary human Tcells. In vitro data demonstrated specific activation, proliferation,and killing of CD37 positive cancer cells by anti-CD37 CAR T cells.

CD37 is detectable in certain cancer-related cell lines (FIG. 34 ).Anti-CD37 CARs were constructed (FIG. 35 ) and expressed in T cells(FIG. 36 ). Expansion of the T cells was measured (FIG. 37 ), and theirability to lyse target cancer cells demonstrated (FIG. 38 ). Anti-CD37demonstrated proliferation in response to CD37 stimulation (FIG. 40 ).

Anti-CD37 CAR Ts can be administered to mice to demonstrate tumorclearance in vivo (FIG. 41 ).

Example 6

Anti-CD37 chimeric antigen receptor T cells: a new potential therapeuticoption for B-cell malignancies.

CD37 is a tetraspanin expressed on mature B cells but absent on earlyprogenitors or terminally differentiated plasma cells. CD37 is highlyexpressed on malignant B cells in non-Hodgkin lymphomas (NHL), includingmantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), Burkitt lymphoma and B-cell chroniclymphocytic leukemia (CLL); thus, CD37 represents a promising target forB-cell malignancies, particularly for variants that escape existingtherapies targeting the common B cell antigens CD19 and CD20.

Described herein is an anti-CD37 CAR (CAR-37) for the treatment ofB-cell malignancies. Specifically, described herein is asecond-generation CAR, encoded by a lentiviral vector and bearing a4-1BB costimulatory domain. Two different orientations of a humanizedmurine antibody-derived single-chain variable fragment (V_(L)-V_(H) orV_(H)—V_(L)) were tested and a and pre-clinical data panel is providedherein.

Results

In vitro cytotoxic activity of CAR T-37 cells was evaluated byco-culturing CAR T-37 cells with CD37-expressing human tumor cell lines(RAJI, OSU-CLL and JEKO-1) at different effector to target ratios.CD37-directed CAR T cells demonstrated antigen-specific activation,proliferation, cytokine production, and cytotoxic activity in vitro inmultiple models of B cell malignancy. Next, the anti-lymphoma efficacywas assessed in vivo in a mantle cell lymphoma model. CAR-37 treatmenteliminated the tumor cells within 2 weeks, and mice maintained durableremissions. CAR T cells were detectable in the blood of mice after 7days of injection. Ongoing studies are evaluating the long termpersistence of CAR T cells in mice.

Discussion

Taken together these results demonstrate that T cells expressinganti-CD37 CAR have substantial activity in vitro and in vivo against Bcell malignancies. These findings indicated that CD37-CAR T cells are anovel potential therapeutic agent for the treatment of patients withCD37 expressing tumors.

Example 7

CD37 is highly expressed in B cell malignancies (FIG. 43 ). Anti-CD37CARs were constructed using the humanized anti-CD37 mAb BI836826(Boehringer Ingelheim) and cloned into a lentivirus vector.Intracellular domains are 41BB (CD137) ICD linked to CD3zeta.

The anti-CD37 CAR T cells were demonstrated to expand upon stimulation(FIG. 44 ) and activate (FIG. 45 ). Anti-CD37 CAR Ts expanded uponstimulation with the target antigen (FIG. 46 ).

The anti-CD37 CAR Ts were demonstrated to lyse tumor cells in vitro(FIG. 47 ) and the production of various cytokines was measured (FIG. 48). In vivo efficacy of the anti-CD37 CAR Ts was demonstrated (FIGS. 49,50 ).

Example 8

scFv Sequences

BCMA scFv (SEQ ID NO: 1) comprises a VH chain (amino acids 1-121 (SEQ IDNO: 2)), a linker region (amino acids 122-141 (SEQ ID NO: 3)), and a VLchain (amino acids 142-249 (SEQ ID NO: 4)).

(SEQ ID NO: 1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKL PWTFGQGTKLEIKR

VH chain (SEQ ID NO: 2 (amino acids 1-121 of SEQ ID NO: 1))

(SEQ ID NO: 2) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS

Linker region (SEQ ID NO: 3 (amino acids 122-141 of SEQ ID NO: 1))

(SEQ ID NO: 3) GGGGSGGGGSGGGGSGGGGS

VL chain (SEQ ID NO: 4 (amino acids 142-249 SEQ ID NO: 1))

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKL PWTFGQGTKLEIKR

CD37 scFv VH-VL (SEQ ID NO: 5) comprises a VH chain (amino acids 1-116(SEQ ID NO: 6)), a linker region (amino acids 117-136 (SEQ ID NO: 7)),and a VL chain (amino acids 137-244 (SEQ ID NO: 8)).

(SEQ ID NO: 5) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEFVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFG QGTKVEIKR

VH chain (SEQ ID NO: 6 (amino acids 1-116 of SEQ ID NO: 5)

(SEQ ID NO: 6) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVY YCARSVGPMDYWGQGTLVTVSS

Linker region (SEQ ID NO: 7 (amino acids 117-136 of SEQ ID NO: 5)

(SEQ ID NO: 7) GGGGSGGGGSGGGGSGGGGS

VL chain (SEQ ID NO: 8 (amino acids 137-244 SEQ ID NO: 5)

(SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDN PWTFGQGTKVEIKR

CD37 scFv VL-VH (SEQ ID NO: 9) comprises a VL chain (amino acids 1-108(SEQ ID NO: 10)), a linker region (amino acids 109-128 (SEQ ID NO: 11)),and a VH chain (amino acids 129-244 (SEQ ID NO: 12)).

(SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWG QGTLVTVSS

VL chain (SEQ ID NO: 10 (amino acids 1-108 of SEQ ID NO: 9))

(SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDN PWTFGQGTKVEIKR

Linker region (SEQ ID NO: 11 (amino acids 109-128 of SEQ ID NO: 9))

(SEQ ID NO: 11) GGGGSGGGGSGGGGSGGGGS

VH chain (SEQ ID NO: 12 (amino acids 129-244 SEQ ID NO: 9))

(SEQ ID NO: 12) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSV GPMDYWGQGTLVTVSS

Example 9

ITAM Sequences

CD3ζ-ITAM 3 (SEQ ID NO: 13)

(SEQ ID NO: 13) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

CD3ζ-mutITAM 1 (SEQ ID NO: 14). As described herein, certain residuesare mutated in CD3ζ-ITAM 3 to inhibit the function of CD3ζ-ITAM 3,namely: Y21 and Y32. The locations of these residues are depicted belowwith bold type.

(SEQ ID NO: 14) RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

CD3ζ-mutITAM1 and mutITAM2 (SEQ ID NO: 33). As described herein, certainresidues are mutated in CD3ζ-ITAM 3 to inhibit the function of CD3ζ-ITAM3, namely: Y21, Y32, Y59 and Y71. The locations of these residues aredepicted below with bold type.

(SEQ ID NO: 33) RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

CD3ζ-mutITAM1 and mutITAM3 (SEQ ID NO: 34). As described herein, certainresidues are mutated in CD3ζ-ITAM 3 to inhibit the function of CD3ζ-ITAM3, namely: Y21, Y32, Y90 and Y100. The locations of these residues aredepicted below with bold type.

(SEQ ID NO: 34) RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDT FDALHMQALPPR

Example 10

CAR Sequences

pMGH8-CD8Leader/anti-BCMA/CD8hinge+TM/4-1BB/CD3ζ (SEQ ID NO: 15)comprising CD8 leader sequence (amino acids 1-21 (SEQ ID NO: 16));anti-BCMA (amino acids 22-270 (SEQ ID NO: 17)); CD8 hinge and TM domain(amino acids 271-339 (SEQ ID NO: 18)); 4-1BB (amino acids 340-381 (SEQID NO: 19)); and CD3ζ (amino acids 382-493 (SEQ ID NO: 20)).

(SEQ ID NO: 15) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD8 leader sequence (SEQ ID NO: 16 (amino acids 1-21 of SEQ ID NO: 15))

(SEQ ID NO: 16) MALPVTALLLPLALLLHAARP

anti-BCMA (SEQ ID NO: 17 (amino acids 22-270 of SEQ ID NO: 15))

(SEQ ID NO: 17) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR

CD8 hinge and TM domain (SEQ ID NO: 18 (amino acids 271-339 of SEQ IDNO: 15))

(SEQ ID NO: 18) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

4-1BB (SEQ ID NO: 19 (amino acids 340-381 of SEQ ID NO: 15))

(SEQ ID NO: 19) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3ζ (SEQ ID NO: 20 (amino acids 382-493 of SEQ ID NO: 15))

(SEQ ID NO: 20) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

pMGH8-CD8Leader/anti-CD37 H-L/CD8hinge+TM/4-1BB/CD3ζ (SEQ ID NO: 21)comprising CD8 leader sequence (amino acids 1-21 (SEQ ID NO: 22));anti-CD37 H-L (amino acids 22-265 (SEQ ID NO: 23)); CD8 hinge and TMdomain (amino acids 266-334 (SEQ ID NO: 24)); 4-1BB (amino acids 335-376(SEQ ID NO: 25)); and CD3ζ(amino acids 377-488 (SEQ ID NO: 26)).

(SEQ ID NO: 21) MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD8 leader sequence (SEQ ID NO: 22 (amino acids 1-21 of SEQ ID NO: 21))

(SEQ ID NO: 22) MALPVTALLLPLALLLHAARP

anti-CD37 H-L (SEQ ID NO: 23 (amino acids 22-265 of SEQ ID NO: 21))

(SEQ ID NO: 23) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS

CD8 hinge and TM domain SEQ ID NO: 24 ((amino acids 266-334 of SEQ IDNO: 21))

(SEQ ID NO: 24) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

4-1BB (SEQ ID NO: 25 (amino acids 335-376 of SEQ ID NO: 21))

(SEQ ID NO: 25) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3ζ (SEQ ID NO: 26 (amino acids 377-488 of SEQ ID NO: 21))

(SEQ ID NO: 26) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

pMGH8-CD8Leader/anti-CD37 L-H/CD8hinge+TM/4-1BB/CD3ζ (SEQ ID NO: 27)comprising CD8 leader sequence (amino acids 1-21 (SEQ ID NO: 28));anti-CD37 L-H (amino acids 22-265 (SEQ ID NO: 29)); CD8 hinge and TMdomain (amino acids 266-334 (SEQ ID NO: 30)); 4-1BB (amino acids 335-376(SEQ ID NO: 31)); and CD3ζ (amino acids 377-488 (SEQ ID NO: 32)).

(SEQ ID NO: 27) MALPVTALLLPLALLLHAARPAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD8 leader sequence (SEQ ID NO: 28 (amino acids 1-21 of SEQ ID NO: 27))

(SEQ ID NO: 28) MALPVTALLLPLALLLHAARP

anti-CD37 L-H (SEQ ID NO: 29 (amino acids 22-265 of SEQ ID NO: 27))

(SEQ ID NO: 29) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR

CD8 hinge and TM domain SEQ ID NO: 30 ((amino acids 266-334 of SEQ IDNO: 27))

(SEQ ID NO: 30) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

4-1BB (SEQ ID NO: 31 (amino acids 335-376 of SEQ ID NO: 27))

(SEQ ID NO: 31) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3ζ (SEQ ID NO: 32 (amino acids 377-488 of SEQ ID NO: 27))

(SEQ ID NO: 32) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

anti-BCMA CAR-mutITAM1 (SEQ ID NO: 35). As described herein, certainresidues are mutated in this anti-BCMA CAR to inhibit the function ofCD3ζ-ITAM, namely: Y402 and Y413 of SEQ ID NO: 35. The locations ofthese residues are depicted below with bold type.

(SEQ ID NO: 35) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

anti-BCMA CAR-mutITAM2 (SEQ ID NO: 36). As described herein, certainresidues are mutated in this anti-BCMA CAR to inhibit the function ofCD3ζ-ITAM, namely: Y440 and Y452 of SEQ ID NO: 36. The locations ofthese residues are depicted below with bold type.

(SEQ ID NO: 36) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

anti-BCMA CAR-mutITAM3 (SEQ ID NO: 37). As described herein, certainresidues are mutated in this anti-BCMA CAR to inhibit the function ofCD3ζ-ITAM, namely: Y471 and Y482 of SEQ ID NO: 37. The locations ofthese residues are depicted below with bold type.

(SEQ ID NO: 37) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

anti-BCMA CAR-mutITAM1 and mutITAM3 (SEQ ID NO: 38). As describedherein, certain residues are mutated in this anti-BCMA CAR to inhibitthe function of CD3ζ-ITAM, namely: Y402, Y413, Y471 and Y482 of SEQ IDNO: 38. The locations of these residues are depicted below with boldtype.

(SEQ ID NO: 38) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

CD3 eta CAR (SEQ ID NO: 39)

(SEQ ID NO: 39) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQDSHFQAVPVQEKKKRLRRAPWRAFAQPQRLKHPAEQPIVRQCLQRPPLCGVLGPVQQQLPPSLGPVLSPHSDPGWCRVDDGGDGVFSGGGGEGRGSLLTCGDVEENPGPRMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQ YERAEGRHSTGGMDELYK

CD3 theta CAR (SEQ ID NO: 40)

(SEQ ID NO: 40) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQDSHFQAVPVQEKKKRLRRAPWRAFAQPQRLKHRNNELPDSLEPIYKNIWNKTFIGESGGGGEGRGSLLTCGDVEENPGPRMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

Example 11

Anti-CD37 CAR-T Cells Effective Against B Cell Malignancies

CD37 is a tetraspanin expressed on mature B cells but absent on earlyprogenitors or terminally differentiated plasma cells. It is highlyexpressed on malignant B cells and it represents a promising target forB-cell malignancies, particularly for variants that escape existingtherapies targeting the common B-cell antigens CD19 and CD20. Wedesigned the first anti-CD37 CAR (CAR-37) for the treatment of B-cellmalignancies. In vitro cytotoxic activity of CART-37 cells was evaluatedby co-culturing CART-37 cells with CD 37-expressing human tumor celllines at different effector to target ratios. CD37-directed CAR T cellsdemonstrated antigen-specific proliferation, cytokine production, andcytotoxic activity in vitro in multiple models of B-cell malignancy. Weassessed the anti-lymphoma efficacy in vivo in a mantle cell lymphomamodel. CAR-37 treatment eliminated the tumor cells within 2 weeks, andmice maintained durable remissions. Together these results show that Tcells expressing anti-CD37 CAR have substantial activity in vitro and invivo against B-cell malignancies. These findings indicated that CD37-CART cells are a novel potential therapeutic agent for the treatment ofpatients with CD37-expressing tumors.

In normal tissues, CD37 expression is restricted to lymphoid organs(FIG. 51A). However, it is highly expressed on B-cell leukemia andlymphoma cells (FIG. 51B).

CAR-37 T cells are able to proliferate and expand ex-vivo (FIGS.53A-53D).

T cells expressing anti-CD37 CAR have substantial in vitro activityagainst mantle cell lymphoma, Burkitt lymphoma and B-cell lymphoblasticleukemia tumor cells (FIGS. 54A-54D).

T cells expressing anti-CD37 CAR have a strong anti-tumor activity in amantle cell lymphoma model in vivo (FIGS. 56A-56C)

Example 12

CD37 and CD19 Expression in Human Tumor Cell Lines

The expression of CD37 and CD19 in human tumor cell lines was evaluated(FIG. 58 ). The top panel of FIG. 57 reproduces the data shown in FIG.51A, and shows that CD37 is highly expressed in non-Hodgkin lymphomasincluding MCL (JEKO-1), Burkitt lymphoma (RAJI), and B-cell chroniclymphocytic leukemia (OSU-CLL), but is absent in the ALL cell lineNALM6. The bottom panel of FIG. 57 shows the expression of CD37 and CD19in the MCL patient-derived xenograft (PDX) lines PDX_44685, PDX_98848,and PDX_96069, as well as the percent expression of CD19 and CD37 in thePDX cell lines. The MCL PDX cell lines expressed both CD37 and CD19.

Example 13

Anti-CD37 CAR-T Cells are Effective Against MCL PDX Tumors

The efficacy of anti-CD37 CAR-T cells against MCL PDX cells wasevaluated in vivo. FIG. 58A shows an experimental schematic; NOD/SCIDmice were injected i.v. with 1×10⁶ PDX_98848 cells. On day 0, micereceived 3×10⁶ control T cells (UTD), CAR-37 H-L, or CAR-19. Tumorgrowth was evaluated by BLI on day 3, day 7, day 10, day 14, day 17, day21, and day 35. Representative bioluminescent images of the PDX growthover time is shown in FIG. 58B. T cells expressing anti-CD37 CAR havestrong anti-tumor activity against MCL PDX in vivo (FIGS. 58A-58C).

Example 14

CD37 Expression in Peripheral T Cell Lymphoma (PTCL)

The expression of CD37 was evaluated in PTCL cell lines, including HUT78(cutaneous T-cell lymphoma (CTCL)) and FEPD (anaplastic large celllymphoma (ALCL)) (FIG. 59A). CD37 was expressed in both cell lines.Expression of the early activation marker CD69 was examined after CARstimulation (FIGS. 59B and 59C).

Example 15

Anti-CD37 CAR-T Cells have In Vitro Cytotoxic Activity Against PTCL CellLines

The in vitro cytotoxic activity of anti-CD37 CAR-T cells against PTCLlines was evaluated (FIG. 60 ). T-cells expressing anti-CD37 CAR havesubstantial in vitro activity against PTCL lines, including CTCL andALCL tumor models (FIG. 60 ). These findings demonstrated that CD37-CART cells are useful as therapeutic agents for the treatment of patientswith PTCL, including CTCL and ALCL.

Example 16

CD37 Expression in AML

CD37 expression has been detected in AML samples (Pereira et al. Mol.Cancer Ther. 14(7):1650-1660, 2015). The expression of CD37 in AML celllines was evaluated in the AML cell lines TF1, MOLM13, and THP by flowcytometry (FIG. 61 ). All three AML cell lines expressed CD37 (FIG. 61). These findings demonstrated that CD37-CAR T cells are useful astherapeutic agents for the treatment of patients with AML.

What is claimed herein is:
 1. A chimeric antigen receptor (CAR)polypeptide comprising: a) an extracellular domain comprising atarget-binding sequence that binds to BCMA, CD37 or CD19; b) atransmembrane domain; c) a co-stimulatory domain; and d) a T cellintracellular signaling domain, comprising a mutation of a tyrosineresidue in an immunoreceptor tyrosine-based activation motif (ITAM) II.2. The CAR polypeptide of claim 1, wherein the transmembrane domain isthe transmembrane domain from CD8 or 4-1BB.
 3. The CAR polypeptide ofclaim 1, wherein the intracellular signaling domain comprises a CD3ζITAM3 sequence of SEQ ID NO:
 33. 4. The CAR polypeptide of claim 1,wherein the intracellular signaling domain comprises a CD3ζ ITAM3sequence comprising a mutation of a tyrosine residue in ITAM II relativeto the CD3ζ ITAM3 sequence of SEQ ID NO:
 13. 5. The CAR polypeptide ofclaim 1, wherein the T-cell intracellular signaling domain is theintracellular signaling domain of CD3ε or CD3θ.
 6. The CAR polypeptideof claim 1, wherein the target-binding sequence comprises a ligand ofthe target or an antibody reagent that specifically binds the target. 7.The CAR polypeptide of claim 1, wherein the target is B cell maturationantigen (BCMA).
 8. The CAR polypeptide of claim 1, wherein the target isCD37.
 9. The CAR polypeptide of claim 1, wherein the target-bindingsequence has the sequence of SEQ ID NO:
 1. 10. A chimeric antigenreceptor (CAR) polypeptide comprising: a) an extracellular domaincomprising a BCMA-binding sequence; b) a transmembrane domain of CD8; c)a co-stimulatory domain of a 4-1BB; and d) a T cell intracellularsignaling domain comprising a mutation of a tyrosine residue in animmunoreceptor tyrosine-based activation motif (ITAM) II.
 11. The CARpolypeptide of claim 10, wherein the BCMA-binding sequence comprises aligand of BCMA or an antibody reagent that specifically binds BCMA. 12.The CAR polypeptide of claim 11, wherein the antibody reagent comprisesthe sequence of SEQ ID NO:
 1. 13. The CAR polypeptide of claim 10,wherein the intracellular signaling domain comprises a CD3ζ ITAM3sequence comprising a mutation of a tyrosine residue in ITAM II relativeto the CD3ζ ITAM3 sequence of SEQ ID NO:
 13. 14. A chimeric antigenreceptor (CAR) polypeptide comprising: a) an extracellular domaincomprising a CD37-binding sequence; b) a transmembrane domain of CD8; c)a co-stimulatory domain of a 4-1BB; and d) a T cell intracellularsignaling domain comprising a mutation of a tyrosine residue in animmunoreceptor tyrosine-based activation motif (ITAM) II.
 15. The CARpolypeptide of claim 14, wherein the CD37-binding sequence comprises aligand of CD37 or an antibody reagent that specifically binds CD37. 16.The CAR polypeptide of claim 15, wherein the antibody reagent comprisesa scFv.
 17. The CAR polypeptide of claim 14, wherein the intracellularsignaling domain comprises a CD3ζ ITAM3 sequence comprising a mutationof a tyrosine residue in ITAM II relative to the CD3ζ ITAM3 sequence ofSEQ ID NO:
 13. 18. The CAR polypeptide of claim 4, wherein the CD3ζITAM3 sequence comprises a substitution mutation at amino acidspositions relative to Y59 and Y71 of SEQ ID NO:
 13. 19. The CARpolypeptide of claim 18, wherein CD3ζ ITAM3 sequence comprises aphenylalanine at amino acid positions Y59 and Y71 of SEQ ID NO:
 13. 20.The CAR polypeptide of claim 18, wherein the CD3ζ ITAM3 sequencecomprises a phenylalanine at amino acid positions Y90 and Y100 of SEQ IDNO:
 13. 21. The CAR polypeptide of claim 20, wherein the extracellulardomain comprises a target-binding sequence that binds to CD19.
 22. TheCAR polypeptide of claim 21, wherein the transmembrane domain is a CD28transmembrane domain.
 23. A nucleic acid sequence comprising apolynucleotide encoding the CAR polypeptide of claim
 1. 24. A mammaliancell comprising the CAR polypeptide of claim
 1. 25. The mammalian cellof claim 24, wherein the mammalian cell is a T cell.
 26. The mammaliancell of claim 24, wherein the mammalian cell is a human cell.
 27. Acomposition comprising the CAR polypeptide of claim
 1. 28. A compositioncomprising the T cell of claim 25.